IRLF 


810UKY  LIBRARY 


A  LABORATORY  MANUAL 
OF  BIOLOGICAL  CHEMISTRY 


LABORATORY  MANUAL 
OF  BIOLOGICAL  CHEMISTRY 


LABORATORY  MANUAL  OF 
BIOLOGICAL  CHEMISTRY 

WITH   SUPPLEMENT 


BY 
OTTO  FOLIN 

Hamilton  Kuhn  Professor  of  Biological  Chemistry  in  Harvard  Medical  School 


NEW  YORK  AND  LONDON 

D.  APPLETON  AND   COMPANY 

1916 


'57? 


BIOLOGY 
LIBRARY 

• 


COPYRIGHT,  1916,  BT 
D.  APPLETON  AND  COMPANY 


Printed  in  the  United  States  of  America 


PREFACE 

This  manual  of  biological  chemistry  for  medical  students  in 
Harvard  Medical  School  has  been  revised  annually  for  the  past 
seven  years,  and  it  is  believed  now  to  meet  our  needs  sufficiently 
well  to  warrant  publication. 

For  many  years  I  have  been  interested  in  the  development  of 
analytical  methods  applicable  to  metabolism  investigations.  The 
most  serviceable  of  my  older  methods  and  some  of  the  newer 
methods  have  been  taught  to  our  medical  students ;  these  are  de- 
scribed in  the  main  body  of  the  manual.  Others  not  heretofore 
included  have  been  incorporated  in  the  supplement,  so  that  nearly 
all  the  newer  methods  devised  in  the  department  are  now  de- 
scribed in  this  manual. 

In  connection  with  the  revisions  referred  to  above  I  am  in- 
debted for  valuable  help  to  W.  R.  Bloor,  W.  Denis,  C.  J.  Farmer, 
L.  J.  Morris,  F.  B.  Kingsbury,  F.  S.  Hammett,  R.  D.  Bell,  and 
C.  H.  Fiske,  as  well  as  to  my  older  friend,  P.  A.  Shaffer. 

OTTO  FOLIN. 
BOSTON. 


343694 


TABLE  OF   CONTENTS 

CHAPTER  PAGE 

I     ACIDIMETRY,    ALKALIMETRY,    NITROGEN    DETERMINA- 
TIONS     1-23 

II  CATALYSIS,  CATALYZERS,  FERMENTS       ....  25-29 

III  FATS 31-37 

IV  CARBOHYDRATES 39~59 

V  PROTEINS          .        . 61-77 

VI  URINE  ANALYSIS   AND  METABOLISM     ....    79-107 

VII  BLOOD 109-111 

VIII  MILK 113-115 

IX  BONE 117 

X  BILE r     .  119-121 


SUPPLEMENT 


URINE 
BLOOD 


-  125-157 
.  159-185 


LABORATORY   MANUAL   OF  BIO- 
LOGICAL CHEMISTRY 

PART  I 
ACIDIMETRY,  ALKALIMETRY,  NITROGEN  DETERMINATION 

Equivalent  and  Normal  Solutions. — Since  the  molecular  weight 
of  sodium  hydroxid  (NaOH)  is  40  and  that  of  hydrochloric  acid 
(HC1)  is  36.46,  it  follows  that  40  g.  of  the  former  contain  the 
same  number  of  molecules  as  36.46  g.  of  the  latter.  If  40  g.  of 
sodium  hydroxid  and  36.46  g.  of  hydrochloric  acid  are  each  dis- 
solved in  pure  water  sufficient  to  make  one  liter  of  solution,  each 
liter  will  contain  the  same  number  of  dissolved  molecules. 

It  will  take  a  little  less  than  one  liter  of  water  to  make  a  liter  of 
solution  because  the  dissolved  substance  takes  up  some  space.  A  nor- 
mal sodium  hydroxid  solution  contains  four  per  cent,  of  sodium 
hydroxid.  By  per  cent,  in  the  case  of  solutions  is  usually  meant  the 
amount  of  substance  present  in  100  c.c.  of  solution. 

Mixing  equal  volumes  of  two  such  solutions  is  therefore  the  same 
as  bringing  together  practically  the  same  number  of  the  two  kinds 
of  molecules,  and  the  result  is  the  instantaneous  and  essentially 
complete  transformation  into  sodium  chlorid  (and  water). 

X  NaOH  +  X  HC1  =  X  NaCl  +  X  H2O 

If  either  or  both  of  the  solutions  should  first  be  diluted  with  a 
considerable  bulk  of  pure  water,  the  result  on  mixing  the  two 
would  be  the  same,  for  the  extra  amount  of  water  present  takes 
no  part  in  the  reaction  (except  to  the  extent  of  absorbing  a  part 
of  the  heat  set  free). 

The  two  solutions  are  equivalent.  They  also  happen  to  be  nor- 
mal solutions.  The  hydrochloric  acid  is  normal  because  it  con- 
tains i  g.  of  active  or  replaceable  hydrogen  per  liter  of  solution, 

i 


and  not  because  it  contains  the  same  number  of  grams  of  HQ 
per  liter  as  there  are  units  in  the  molecular  weight.  The  sodium 
hydroxid  solution  is  normal  because  it  is  equivalent  to  a  solution 
containing  one  gram  of  replaceable  hydrogen  per  liter. 

The  molecular  weight  of  sulphuric  acid  is  98.  A  sulphuric  acid 
solution  containing  exactly  98  g.  per  liter  contains  therefore  the 
same  number  of  molecules  per  unit  volume  as  the  sodium  hy- 
droxid solution  containing  40  g.  per  liter.  But  one  molecule  of 
sulphuric  acid  requires  two  molecules  of  sodium  hydroxid  for  the 
formation  of  the  neutral  salt,  sodium  sulphate,  because  the  sul- 
phuric acid  molecule  has  two  replaceable  hydrogen  atoms.  The 
solutions  are  not  equivalent,  for  the  sulphuric  acid  contains  2  g. 
active  hydrogen  per  liter.  It  is  exactly  twice  as  strong  as  the 
sodic  hydrate  solution ;  it  is  a  2  normal  solution. 

On  the  basis  of  the  above  description  of  what  constitutes  a  normal 
solution,  calculate  the  number  of  grams  per  liter  in  tenth  normal  sul- 
phuric acid  (.iN  H2SO4),  fifth  normal  hydrochloric  acid  (.2NHC1), 
half  normal  oxalic  acid  (.5N  C2H204,  2H2O),  fourth  normal  acetic 
acid  (.25N  CH3COOH),  half  normal  sodic  hydrate  (.sN  NaOH), 
twentieth  normal  barium  hydrate  (.05N  Ba(OH)2),  fifth  normal 
ammonium  hydrate  (.2N  NH4OH). 

Atomic  weights  of  some  of  the  more  important  elements:  Arsenic 
(As)  74.96,  Barium  (Ba)  137.37,  Bromin  (Br)  79.92,  Calcium  (Ca) 
40.09,  Carbon  (C)  12,  Chlorin  (Cl)  35.46,  Copper  (Cu)  63.57, 
Hydrogen  (H)  1.008,  lodin  (I)  126.92,  Iron  (Fe)  55.85,  Lead  (Pb) 
207.1,  Magnesium  (Mg)  24.32,  Manganese  (Mn)  54.93,  Mercury 
(Hg)  200.6,  Nitrogen  (N)  14.01,  Oxygen  (O)  16,  Phosphorus  (P) 
31.04,  Potassium  (K)  39.1,  Sulphur  (S)  32.07,  Tin  (Sn)  119, 
Tungsten  (Wo)  184,  Uranium  (U)  238.5,  Zinc  (Zn)  65.37. 

The  same  description  of  normal  solutions  applies  to  other  sub- 
stances than  acids  and  alkalis,  as  for  example,  reducing  and  oxi- 
dizing substances  such  as  potassium  permanganate,  potassium  bi- 
chromate, iodin,  cupric  hydrate,  stannous  chlorid.  A  normal  solu- 
tion is  here  one  capable  of  liberating  I  g.  of  reducing  hydrogen 
(or  of  giving  off  exactly  sufficient  oxygen  to  oxidize  one  gram  of 
hydrogen)  per  liter.  Potassium  permanganate,  for  example,  in 
the  presence  of  sulphuric  acid  and  some  easily  oxidizable  sub- 
stance is  decomposed  as  follows : 

2K  MnO4  +  3H2SO4  =  K2SO4  +  2Mn  SO4  +  3H2O  +  50 
+  2  MnSO4 

3 


As  the  two  permanganate  molecules  liberate  oxygen  enough  for 
ten  hydrogen  atoms  it  takes  only  one  fiftieth  of  the  molecular 
weight  expressed  in  grams  (3.156  g.)  to  make  one  liter  of  tenth 
normal  solution. 

The  calculation  of  what  constitutes  normal  or  equivalent  solu- 
tions of  any  reagent  is  not  very  difficult  provided  the  equation 
representing  the  cherrfical  reaction  involved  is  thoroughly  clear. 

To  determine  whether  a  given  unknown  solution  is  acid  or 
alkaline  it  is  usually  sufficient  to  -dip  a  piece  of  delicate  violet 
colored  litmus  paper  into  it.  (If  the  solution  is  acid  the  test 
paper  turns  red;  if  alkaline  it  turns  blue.)  Litmus,  the  substance 
with  which  the  paper  has  been  impregnated,  is  a  complex  organic 
product,  and  is  one  of  the  most  familiar  representatives  of  a 
most  useful  class  of  organic  compounds  which  are  so  sensitive 
to  acids  or  alkalis,  or  both,  that  they  clearly  and  unmistakably  in- 
dicate the  presence  of  free  acid  or  alkali  even  when  the  amounts 
present  are  so  small  as  to  be  practically  unweighable.  By  means 
of  such  indicators  and  accurate  measuring  instruments  (measur- 
ing flasks,  burets,  and  pipets),  it  becomes  a  simple  matter  to 
determine  (by  titration)  the  relative  concentration  or  equivalence 
of  acid  and  alkaline  solutions.  By  their  help  it  is  possible  to  pre- 
pare with  very  little  labor  normal  or  tenth  normal  solutions,  even 
of  acids  or  alkalis  which  cannot  be  weighed  on  the  balance,  as  for 
example,  hydrochloric  acid  and  ammonia,  both  of  which  are  gases. 
Before  this  can  be  done  we  must,  however,  possess  one  normal 
or  standard  solution  prepared  from  some  substance  which  can 
be  weighed. 

Volumetric  analysis  consists  of  measuring  the  value  of  an  un- 
known solution  in  terms  of  another  the  value  of  which  is  known 
(titration).  The  known  solution  is  prepared  directly  or  indirectly 
by  the  help  of  the  analytical  balance,  and  the  first  step  in.  any 
kind  of  volumetric  analysis  is  the  preparation  of  the  standard 
solution  by  means  of  which  the  values  of  others  are  to  be  deter- 
mined. 

Every  student  who  has  had  no  experience  in  the  use  of  the  ana- 
lytical balance  must  consult  the  instructor  before  proceeding.  He 
should  also  ask  for  instruction  as  to  the  proper  use  of  measuring 
flasks,  pipets,  and  burets  before  using  them.  He  must  particularly 
learn  when  the  presence  of  unmeasured  quantities  of  water  does  not 
interfere  with  the  accuracy  of  the  work  and  when  a  single  drop  of 

5 


unmeasured  water  introduces  a  perceptible  error.  (See  Button's 
Volumetric  Analysis,  Part  i — "Instruments  and  Apparatus.") 

All  the  common  mineral  acids  and  strong  alkalis  contain  so 
much  water  that  it  is  in  practice  not  feasible  to  weigh  out  with 
sufficient  accuracy  the  theoretical  quantity  required  for  a  standard 
solution  of  acid  or  alkali.  The  carbonates  of  sodium  or  calcium 
(or  the  carbonates  of  sodium  or  potassium,  obtained  by  ignition 
of  the  corresponding  oxalates)  give  exceedingly  accurate  results. 
Oxalic  acid  is  very  serviceable  as  starting  material  for  the  prepa- 
ration of  standardized  solutions  of  acids  and  alkalis  if  it  is  pure 
and  has  lost  none  of  its  water  of  crystallization. 

1.  Preparation  of  .5N  Oxalic  Acid  (500  G.C.)  — The  usefulness 
of  oxalic  acid  as  a  starting  point  for  the  preparation  of  standard 
acids  and  alkalis  is  due  entirely  to  the  fact  that  it  can  be  obtained 
chemically  pure  and  in  condition  suitable  for  direct  weighing.  Oxalic 
acid  is,  however,  not  a  strong  enough  acid  to  titrate  well  with  all 
the  common  indicators,  and  it  is  therefore  not  serviceable  for  acidi- 
metric  titrations  in  general.  But  by  means  of  oxalic  acid  and  with 
phenolphthalein  as  indicator,  standard  solutions  of  a  strong  alkali, 
like  caustic  soda,  can  be  obtained,  and  by  means  of  the  latter  stand- 
ard solutions  of  the  stronger  mineral  acids  can  then  be  prepared. 

The  reason  why  the  strong  acids  and  alkalis  give  more  accurate 
and  reliable  results  is  the  fact  that  the  salts  which  they  form  when 
neutralized  are  not  appreciably  hydrolyzed  by  water  into  acid  and 
base,  as  are  the  corresponding  salts  of  the  weaker  acids  and  bases. 
The  zone  of  neutrality  to  different  indicators  is  therefore  more 
sharply  defined,  and  corresponds  more  nearly  to  the  point  repre- 
sented by  the  presence  of  exactly  equivalent  amounts  of  acid  and 
alkali. 

Weigh  accurately  (to  the  fourth  decimal)  a  small,  clean,  and 
dry  beaker  or  large  crucible.  Then  add  to  the  weights  on  the 
balance  pan  15.7560  g.,  and  add  oxalic  acid  to  the  vessel  on  the 
other  side  until  exact  equilibrium  is  reached.  Dissolve  in  dis- 
tilled water  this  oxalic  acid  without  the  loss  of  a  single  crystal. 
The  acid  dissolves  rather  slowly.  The  solution  is  therefore  best 
made  in  a  beaker  by  the  aid  of  gentle  heating  with  about  250 
c.c.  water.  Transfer  every  drop  of  the  solution  to  a  measuring 
flask  (500  c.c.),  carefully  rinsing  the  last  traces  from  the  beaker 
into  the  flask  by  means  of  successive  small  amounts  of  cold 
distilled  water.  Cool  the  flask  in  running  tap  water  until  the 

7 


contents  of  the  flask  have  reached  the  room  temperature.  (If 
a  thermometer  is  used  it  must  be  rinsed  carefully  before  it  is 
removed  from  the  flask.)  Fill  up  with  water  until  the  lower  side 
of  the  "meniscus"  is  exactly  even  with  the  500  c.c.  mark.  Stop- 
per the  flask,  and  invert  several  times  (30-40)  so  that  the  solu- 
tion is  thoroughly  mixed.  Transfer  to  a  clean,  dry  bottle ;  label 
and  preserve. 

Using  a  strong  base  like  sodium  hydroxid  and  a  sensitive  indi- 
cator like  phenolphthalein  for  the  titration,  it  is  possible  to  obtain 
quite  reliable  and  accurate  results  with  oxalic  acid.  The  volu- 
metric determinations  involved  in  metabolism  studies  and  urine 
analysis  are,  however,  extensively  based  on  titrating  ammonia, 
which  is  a  very  weak  base.  Phenolphthalein,  because  of  its  high 
degree  of  sensitiveness  to  weak  acids  and  its  lack  of  sensitiveness 
to  weak  bases,  is  useless  in  titrations  of  ammonia.  The  oxalic 
acid  and  the  phenolphthalein  are  therefore  used  only  for  the  pur- 
pose of  securing  a  standard  alkali  solution. 

2.  Preparation  of  .5N  Sodic  Hydrate  (1,000  c.c.). — The  sodic 
hydrate  used  for  standard  solutions  must  be  as  free  as  possible 
from  carbonates,  because  otherwise  the  solutions  will  not  have 
the  same  titrating  value  with  all  the  common  indicators.  Sodic 
hydrate  absorbs  rapidly  carbonic  acid  from  the  atmosphere.  The 
carbonic  acid  should  first  be  removed  from  the  alkali,  and  the 
solutions  must  afterwards  be  protected  from  too  much  exposure 
to  the  carbonic  acid  of  the  air.  As  the  carbonates  are  almost 
insoluble  in  very  strong  sodic  hydrate  solutions,  40  per  cent,  solu- 
tions in  which  the  carbonates  have  settled  can  be  used  as  a 
starting  point. 

Transfer  about  80  c.c.  clear  40  per  cent,  sodic  hydrate  solution 
to  a  large  flask  or  bottle,  and  add  1,000-1,200  c.c.  water.  Dissolve 
about  2  g.  barium  hydroxid  in  about  100  c.c.  hot  water,  and  with- 
out filtering  pour  this  solution  into  the  sodic  hydrate  solution. 
Cover  with  a  watch  glass,  and  set  aside  over  night.  By  means  of 
a  siphon  or  compressed  air  remove  the  clear,  supernatant  solution 
to  another  flask. 

To  determine  the  exact  value  of  the  solution  it  is  only  necessary 
to  find  out  how  much  of  it  is  required  for  the  neutralization  of  a 
known  volume  of  the  .$N  oxalic  acid  solution.  With  a  dry  and 
clean  pipet  transfer  25  c.c.  of  the  oxalic  acid  into  a  beaker  or 
flask.  Dilute  it  by  adding  100-150  c.c.  of  water,  and  add  two 

9 


drops  of  the  indicator  (i  per  cent,  alcoholic  solution  of  phenol- 
phthalein). 

Fill  a  dry,  clean  buret  with  the  alkali  and  cover  with  a  test 
tube.  After  adjusting  the  solution  in  the  buret  to  the  zero  mark, 
run  it  into  the  diluted  oxalic  acid,  more  and  more  cautiously 
toward  the  end  until  finally  one  single  drop  produces  a  deep  red 
coloration.  Note  the  volume  of  alkali  required  (to  within  .05 
c.c.).  Repeat  the  titration  until  two  successive  ones  give  exactly 
the  same  figure. 

In  titrating  acids  and  alkalis  the  alkali  must  always  be  run  into 
the  acid  solution,  not  vice  versa. 

From  the  result  obtained  calculate  how  much  of  the'  alkali 
would  be  required  to  neutralize  one  liter  of  the  oxalic  acid  solu- 
tion. By  means  of  a  100  c.c.  pipet  and  a  buret,  transfer  the  re- 
quired amount  of  the  alkali  to  the  liter  measuring  flask,  and  fill 
up  to  the  mark  with  water.  Mix,  transfer  to  a  dry  bottle,  label, 
and  stopper  with  a  rubber  stopper. 

As  a  check  on  the  work  determine  the  concentration  of  an  un- 
known hydrochloric  acid  solution  (furnished),  using  as  indi- 
cator (a)  phenolphthalein  (b)  alizarin  red  (2  drops  I  per  cent, 
aqueous  solution). 

3.  Half    Normal    Hydrochloric    Acid. — Concentrated    hydro- 
chloric acid  is  approximately  10  N  solution  of  HC1.    From  it  pre- 
pare 1,000  c.c.  .5N  solution,  using  the  half  normal  sodium  hy- 
droxid  as  a  standard  and  alizarin  red  as  indicator.     The  most 
convenient  way  is  first  to  prepare  1,200-1,300  c.c.  of  a  solution 
somewhat  stronger  than  half  normal,  and  then,  on  the  basis  of 
titrations  with  the  standard  alkali,  to  dilute  the  required  amount 
with  water  to  one  liter.     One  liter  of  half  normal  hydrochloric 
acid  is  enough  for  all  the  analytical  work  described  in  this  manual. 

Those  who  have  had  experience  in  volumetric  analysis  may  use 
simply  standardized  solutions  instead  of  the  half  normal  in  the  case 
of  hydrochloric  acid  and  in  the  case  of  all  other  standard  solutions, 
but  beginners  should  not  omit  the  preparation  of  a  strictly  half 
normal  acid. 

4.  Tenth  Normal  Acid  and  Alkali. — From  the  half  normal 
stock  solutions  of  hydrochloric  acid  and  sodium  hydroxid,  pre- 

ii 


pare  1,000  c.c.  .iN  hydrochloric  acid  and  .iN  sodium  hydroxid. 
The  solutions  so  obtained  should  be  equivalent. 

5.     Strong  and  Weak  Acids;  the   Use  of  Different  Indicators.* 

(A)  Titrate  25  c.c.  tenth  normal  hydrochloric  acid  (+  Io°  c.c. 
water)  with  the  tenth  normal  alkali,  using  as  indicator  (a)  phenol- 
phthalein  (b)  methyl  orange  (c)  alizarin  red.  Repeat  the  above 
mentioned  three  titrations  in  the  presence  of  10  c.c.  ammonium 
chlorid  solution  (2  per  cent.).  Repeat  the  titration  with  each 
indicator  using  in  place  of  the  hydrochloric  acid  (a)  25  c.c.  .iN 
phosphoric  acid  (b)  25  c.c.  .iN  lactic  acid. 
Record  the  titrations  in  tabular  form : 

Phenolphthalein :       Methyl   orange:  Alizarin   red: 

c.c. — End  point. f       c.c. — End  point. f        c.c.— End  point. f 

HC1: 

HC1  NH4C1 : 
Phosphoric 

acid: 
Lactic  acid: 


(B)  Dilute  (a)  20  c.c.  half  normal  hydrochloric  acid  (b)  20 
c.c.  half  normal  lactic  acid  (supplied)  to  500  c.c.,  making  approx- 
imately .02  N  solutions.  (Measuring  cylinders  are  accurate 
enough  for  the  dilutions  referred  to  here.) 

From  each  .02  normal  solution  prepare  four  250  c.c.  portions 
of  more  dilute  acids,  namely :  .005  N ;  .001  N ;  .0002  N  ;  -.0001  N. 
Arrange  in  a  labeled  series  ten  beakers,  flasks,  or  (small,  white) 
bottles,  similar  in  size  and  shape,  and  transfer  to  each  one  an  ap- 
proximately equal  volume  (50  c.c.)  of  one  of  the  acid  solutions. 
Add  to  each  acid  in  the  series  one  drop  (no  more)  of  "Topfer's 
Reagent"  (.5  per  cent,  dimethylamino-azo-benzol  solution  in  al- 
cohol). For  comparison  add  one  drop  of  the  indicator  to  50  c.c. 
water.  Test  the  acidity  of  each  diluted  acid  with  (a)  congo  red 
paper  (b)  litmus  paper.  Also  taste  each  solution.  (Very  dilute 
acids  are  best  tasted  by  "rinsing  the  mouth"  first  with  distilled 
water  and  then  with  a  liberal  quantity  of  the  acid.) 

*  Consult   Button's  Volumetric  Analysis — "Indicators." 

t  End  point  may  be  designated  as  sharp,  fair,  indistinct,  or  indeterminate 

13 


Record  the  results  of  the  tests  in  the  tabular  spaces  indicated 
below : 


Acid: 

Topfer's  Reagent* 
HC1—  Lactic 

Congo  Red  * 
HC1—  Lactic 

Litmus  * 
HC1—  Lactic 

Sour  Taste  * 
HC1—  Lactic 

.02    N 

.005  N 

.001  N 

.0002  N  ' 

.0001  N 

6.  Special  Test  for  Hydrochloric  Acid. — Gunzberg's  reagent  (2 
g.  phloroglucin  and  i  g.  vanillin  in  100  c.c.  alcohol)  is  very  re- 
liable as  a  means  of  distinguishing  between  hydrochloric  acid  and 
lactic  or  other  organic  acids.     The  reaction  is  best  carried  out 
as  follows: 

Transfer  5-6  drops  of  the  reagent  to  a  shallow  evaporating  dish, 
and  evaporate  to  dryness  over  a  water  bath  consisting  simply  of 
a  beaker  of  boiling  water.  The  alcoholic  solution  spreads  all  over 
the  dish,  leaving  a  thin  coating  of  the  dry  reagent.  By  means 
of  pipets,  or  glass  tubes  drawn  out  like  pipets,  transfer  one  drop 
of  .01  N  hydrochloric  acid  to  one  side  of  the  dish,  and  on  another 
side  deposit  one  drop  of  .1  N  lactic  acid,  and  again  place  the  dish 
on  the  water  bath.  A  purplish  ring  is  quickly  formed  around 
the  hydrochloric  acid  drop  while  the  lactic  acid  remains  colorless. 

Heating  over  the  flame  may  be  substituted  for  the  water  bath, 
but  the  least  overheating  tends  to  obscure  the  reaction  by  charring. 
This  reaction  is  extensively  used  in  the  examination  of  stomach 
contents. 

7.  Special  Test  for  Lactic  Acid. — More  or  less  specific  tests  for 
lactic  acid  are  known  and  are  considered  important  because  of 
the  frequency  with  which  lactic  acid  is  found  in  the  stomach  con- 
tents of  those  suffering  from  carcinoma  of  the  stomach.    A  con- 
venient yet  reliable  method  is  the  following : 

*The  reaction  may  be  designated  as  follows: 
">  ~i      >  ~~~» 

IS 


To  5-10  c.c.  of  .1  N  lactic  acid  (or  filtered  stomach  juice)  in 
a  large  test  tube  add  a  few  drops  (.5  c.c.)  of  normal  hydrochloric 
acid  and  about  10  c.c.  of  ether.  By  cautiously  inverting  the  test 
tube  during  3-4  minutes  (taking  care  to  avoid  explosions  due  to 
expanding  ether  vapors)  the  lactic  acid  is  in  part  taken  up  by  the 
ether.  By  means  of  a  25  c.c.  or  50  c.c.  pipet  and  suction,  remove 
the  lower  ^aqueous  layer  as  completely  as  possible.  Decant  the 
remaining  ether  into  another  test  tube  so  as  to  free  it  from  the 
few  drops  of  aqueous  solution  not  taken  out  by  the  pipet.  Then 
add  to  the  ether  solution  .2  per  cent,  ferric  chlorid  solution,* 
a  little  at  a  time  with  shaking,  until  the  maximum  yellow  color  is 
obtained.  The  amount  of  solution  added  and  the  depth  of  the 
color  obtained  give  a  rough  index  as  to  the  amount  of  lactic  acid 
present. 

8.  Nitrogen  Determination  in  Ammonium  Salts. — The  most 
convenient  and  useful  analysis  of  nitrogenous  products  of  phys- 
iological significance  is  the  determination  of  the  nitrogen.  The 
nitrogen  of  such  products  can  be  split  off  by  hydrolysis  in  the 
form  of  ammonia,  which  can  then  be  determined  by  distillation 
and  subsequent  titration. 

In  a  small  beaker  weigh  (to  the  fourth  decimal)  3-3.5  g.  pure 
ammonium  sulphate.  The  salt  contains  traces  of  water  (.5-1 
per  cent.),  unless  it  has  been  dried  by  heating  1-2  hours  at  about 
110°  C. ;  it  should  be  kept  in  a  desiccator  over  sulphuric  acid. 
Dissolve  the  salt  without  the  loss  of  a  single  crystal  in  a  500  c.c. 
volumetric  flask,  add  1-2  c.c.  concentrated  hydrochloric  acid,  and 
fill  up  to  the  mark  with  water.  The  acid  is  added  to  keep  out 
moulds.  Mix  thoroughly  and  transfer  to  a  dry  bottle,  or  to  a 
bottle  freshly  rinsed  twice  with  about  25  c.c.  of  the  solution. 

This  solution  should  be  stoppered,  labeled,  and  preserved  as  a 
standard  solution.  It  is  used  to  check  up  the  accuracy  of  ammonia 
determinations  and  sulphate  determinations,  and  later  for  colorimetric 
nitrogen  determinations. 

By  means  of  a  pipet  measure  25  c.c.  of  the  ammonium  sul- 
phate solution  into  each  of  two  Jena  flasks  (capacity  about  I 
liter),  and  add  about  350  c.c.  of  water,  a  pinch  of  talcum  powder, 

*  10  c.c.  of  10  per  cent,  ferric  chlorid  solution  in  400-500  c.c.  of  tap 
water. 

17 


and  5  c.c.  of  saturated  sodium  hydroxid  solution.  The  talcum 
powder  is  added  to  prevent  bumping.  The  alkali  is  added  in 
excess  to  set  free  the  ammonia.  In  order  to  avoid  loss  of  the 
volatile  ammonia  the  alkali  must  not  be  added  until  everything 
is  ready  for  the  distillation. 

Beginners  should  always  consult  the  instructor  before  adding  the 
alkali. 

The  lower  part  of  the  condenser  must  dip  into  receivers.  The 
receivers  are  flasks  (capacity,  about  750  c.c.).  In  many  labora- 
tories milk  bottles  are  used  as  receivers.  The  receivers  should 
contain  25  c.c.  .iN  acid  and  two  or  three  drops  of  indicator  (ali- 
zarin red). 

The  indicator  is  added  so  that  if  by  any  chance  the  ammonia 
distilled  over  is  more  than  enough  to  neutralize  the  acid  that  fact 
is  at  once  revealed.  When  this  happens  another  10  or  25  c.c.  of 
.1  N  acid  should  be  added. 

Immediately  after  adding  the  alkali  the  flask  should  be  con- 
nected with  the  condenser.  The  flame  for  the  boiling  should 
then  be  applied  without  delay.  The  distillation  should  be  con- 
tinued until  the  distillate  no  longer  shows  the  faintest  alkaline 
reaction  when  dropped  on  sensitive  litmus  paper.  During  the 
last  few  minutes  the  delivery  tube  should  not  dip  below  the  sur- 
face of  the  liquid  in  the  receiver.  The  time  required  for  the  dis- 
tillation is  usually  about  half  an  hour  from  the  time  the  liquid 
begins  to  boil.  If  a  great  deal  of  water  is  added  to  the  ammonium 
salt  solution,  it  usually  takes  a  little  longer  to  distill  off  all  the 
ammonia.  Brisk  boiling  is  desirable. 

Titrate  the  remaining  uncombined  hydrochloric  acid  in  the  re- 
ceiver, and  from  the  figure  obtained  calculate  the  amount  of  nitro- 
gen recovered  (in  milligrams),  and  compare  with  the  theoretical 
figure  which  the  amount  of  ammonium  sulphate  taken  should 
give. 

In  calculating  the  nitrogen  from  the  titration  figures,  the  amount 
of  acid  combined  with  the  ammonia  can  be  regarded  as  a  tenth 
normal  nitrogen  solution,  each  cubic  centimeter  of  which  accordingly 
represents  1.4,  or  more  accurately  1.401,  milligram  nitrogen.  Ex- 
ample: 25  c.c.  .1  N  HC1  was  the  original  amount  of  acid  in  the 
receiver.  After  the  distillation  the  titration  of  the  distillate  re- 


quired  2.1  c.c.  of  .iN  NaOH.  The  ammonia  had  therefore  neutral- 
ized 25 —  2.1  or  22.9  c.c.  of  the  tenth  normal  acid,  22.9  X  14  =  32.06 
(milligram  nitrogen). 

9.  KjeldahPs  Method  for  Determining  Nitrogen. — In  a  100 
c.c.  volumetric  flask  dissolve  1-1.5  g-  accurately  weighed  urea. 
Add  a  few  drops  (.5-1  c.c.)  concentrated  hydrochloric  acid,  and 
make  the  volume  up  to  100  c.c.,  mix,  and  transfer  to  a  clean  and 
dry  bottle,  label,  and  preserve. 

Pipet  5  c.c.  of  this  solution  into  each  of  two  Kjeldahl  flasks, 
add  15  c.c.  concentrated  ammonia  free  sulphuric  acid  and  2  c.c. 
5  per  cent,  copper  sulphate  solution,  and  boil  30-40  minutes. 

When  organic  substances  are  boiled  with  strong  sulphuric  acid 
both  oxidation  and  hydrolysis  take  place.  The  oxidation  occurs 
at  the  expense  of  the  oxygen  in  the  sulphuric  acid,  and  the  latter  is 
consequently  reduced.  The  sulphurous  fumes  thus  produced  are 
very  irritating  to  the  mucous  membranes  of  the  nose  and  throat. 
The  digestion  must,  therefore,  be  made  in  a  hood  having  a  reason- 
ably good  draft. 

Instead  of  a  hood  a  ''fume  absorber"  can  be  used.  By  the  help 
of  an  ordinary  water  pump  (of  glass)  the  fumes  are  then  partly 
aspirated  directly  into  the  drain  pipes,  and  the  remainder  is  col- 
lected in  the  lower  part  of  the  fume  absorber. 

When  the  urea  is  decomposed  by  means  of  boiling  sulphuric 
acid,  it  is  simply  hydrolyzed  into  carbonic  acid  and  ammonia,  and 
the  ammonia  combines  with  the  sulphuric  acid  to  give  ammonium 
sulphate.  From  this  point  on,  the  nitrogen  determination  is  essen- 
tially the  same  as  the  nitrogen  determination  of  ammonium  sul- 
phate described  above.  There  are,  however,  one  or  two  points 
to  be  noted.  Since  the  ammonium  sulphate  is  now  accompanied 
by  a  relatively  enormous  amount  of  surplus  sulphuric  acid,  a 
great  deal  of  alkali  must  be  added  to  set  free  the  ammonia.  (The 
amount  of  alkali  required  for  this  purpose  should  be  determined 
by  a  rough  titration  of  15  c.c.  concentrated  sulphuric  acid  dis- 
solved in  500-700  c.c.  tap  water — the  saturated  sodium  hydroxid 
solution  being  added  with  a  measuring  cylinder.  Rather  more 
alkali  than  is  indicated  by  the  titration  should  be  used.)  Before 
adding  this  alkali  the  sulphuric  acid  digest  must,  of  course,  be 
cooled  and  diluted  (with  about  300  c.c.  of  water). 

Practices  differ  somewhat  with  regard  to  the  details  in  the  dis- 

21 


dilation  of  Kjeldahl  digestion  mixtures.  Some  use  large  Kjel- 
dahl  flasks  for  the  digestion  (500-750  c.c.),  and  then  use  the 
same  flasks  for  the  distillation.  Others  use  small  Kjeldahl  flasks 
(200  c.c.),  and  pour  the  cooled  acid  into  liter  flasks  containing 
about  200  c.c.  of  water,  rinsing  the  last  traces  of  acid  into  the 
flasks  by  three  or  four  successive  small  quantities  of  water.  The 
latter  procedure  has  this  advantage,  that  there  is  no  danger  from 
explosions  because  the  acid  is  poured  into  water,  and  not  vice 
versa.  Whichever  procedure  is  used,  it  must  not  be  forgotten 
that  much  heat  is  generated  when  concentrated  sulphuric  acid 
and  water  are  mixed,  and  still  more  when  so  much  acid  is  sud- 
denly neutralized  by  the  addition  of  the  strong  alkali.  The  de- 
sirability of  working  understandingly  and  with  reasonable  dis- 
patch when  adding  the  alkali  and  starting  the  distillation  is  clear. 
The  alkali  and  acid  should  not  be  mixed  by  shaking  until  after 
the  flask  is  attached  to  the  condenser. 

The  distillate  is  collected  in  25  c.c.  .1  N  acid  and  50  c.c.  water, 
and  the  nitrogen  recovered  is  determined  and  compared  with 
the  theoretical  figure  as  in  the  ammonium  sulphate  analysis. 

10.  Determination  of  Nitrogen  in  Uric  Acid. — Transfer  50-70 
mg.  of  pure  uric  acid  to  a  clean,  dry  test  tube.  Weigh  the  test 
tube  and  uric  acid  (to  the  fourth  decimal).  Shake  most  of  the 
uric  acid  into  a  dry  Kjeldahl  flask,  and  again  weigh  accurately 
the  empty  test  tube.  The  difference  between  the  two  weighings 
is  the  amount  of  uric  acid  taken.  In  the  same  way,  charge  an- 
other dry  Kjeldahl  flask  with  50-60  mg.  of  uric  acid.  To  each 
flask  add  15  c.c.  of  concentrated  sulphuric  acid  and  2  c.c.  copper 
sulphate  solution,  digest  for  30-40  minutes,  distil,  and  determine 
the  ammonia.  Calculate  as  nitrogen,  and  compare  with  the  the- 
oretical figures. 


PART  II 
CATALYSIS,  CATALYZERS,  FERMEITTS  * 

1.  Hydrogen-ion. — In  each  of  two  test  tubes  place  about  5  c.c. 
of  2  per  cent,  cane  sugar  solution.    To  the  solution  in  one  of  the 
test  tubes  add  a  few  drops  of  concentrated  hydrochloric  acid. 
Boil  the  contents  of  both  for  five  minutes,  or  heat  in  a  beaker  of 
boiling  water  for  ten  minutes.     Cool.     To  the  contents  in  each 
test  tube  add  a  few  drops  of  10  per  cent,  copper  sulphate  solution 
and  a  little  saturated  sodic  hydrate  solution  (2  c.c.).     Heat  the 
alkaline  contents  almost  to  boiling.    Note  and  explain  the  result. 
(Cane  sugar  when  split  by  hydrolysis  yields  reducing  sugars.) 

2.  Hydroxyl-ion. — Fill  a  small  test  tube  up  to  within  about  I  c. 
from  the  top  with  i  per  cent,  tannic  acid  solution.    Add  a  few 
drops  sodic  hydrate  solution,  mix  quickly,  and  let  stand  for  a  few 
minutes. 

3.  Metallic  salts. — To  5  c.c.  of  urine  in  each  of  two  Kjeldahl 
flasks  add  5  c.c.  concentrated  sulphuric  acid.    To  one  add  a  small 
crystal  of  copper  sulphate.    Heat  to  gentle  boiling  for  10  minutes. 
Compare  the  rate  of  disappearance  of  the  brown  color. 

4.  Pepsin. — Prepare  a  pepsin  solution  from  the  mucous  mem- 
brane of  a  pig's  stomach  as  follows :    Strip  off  the  mucous  mem- 
brane from  a  pig's  stomach,  mix  with  300  c.c.  of  approximately 
decinormal   hydrochloric   acid    (the   "concentrated   hydrochloric 
acid"  is  approximately  a  10  N  solution)  in  a  wide-mouth  bottle 
(capacity  900-1,000  C.G.),  and  let  stand  over  night.     Remove  by 
decantation  75  c.c.  of  the  stomach  extract.     To  the  remaining 
mixture  in  the  bottle  add  5-10  c.c.  of  chloroform,  cork  tightly, 
shake  vigorously  for  a  few  seconds,  label,  and  place  in  an  incu- 

*  See  Hammarsten :     "Catalysis"  and  "catalytic  reactions." 

25 


bator.    The  stomach  will  digest  itself  and  give  a  solution  suitable 
for  the  later  study  of  peptones. 

Suspend  a  piece  of  egg  albumin,  or  a  Mett  tube,  in  the  top  of 
each  of  the  following  solutions  : 

(a)  5  c.c.  of  undiluted  gastric  extract, 

(b)  5  c.c.  consisting  of  equal  volumes  of  the  juice  and  .1  N 

hydrochloric  acid, 

(c)  5  c.c.  consisting  of  one  pnrt  of  juice  to  3  parts  .1  N  hydro- 

chloric acid, 

(d)  5  c.c.  of  juice  diluted  as  in  (b)  and  heated  in  a  water  bath 

at  a  temperature  of  75°  for  15  minutes. 

Put  all  the  solutions  in  an  incubator  (the  warm  room)  over 
night.  Note  the  results  and  explain.*  Compare  results  with  those 
of  other  students. 

To  20  c.c.  of  the  pepsin  solution  add  half  a  volume  of  disodic 
phosphate  solution  (10  per  cent.)  and  half  a  volume  of  calcium 
acetate  solution  (10  per  cent).  Filter  off  the  precipitate  and 
wash  once  with  water.  Dissolve  the  precipitate  in  a  minimum 
quantity  of  half  normal  hydrochloric  acid.  Dialyze  over  night. 
Remove  the  liquid  from  the  dialyzing  tube,  test  its  reaction  with 
congo  red,  and  dilute  with  water  or  with  dilute  HC1  (which?) 
to  the  volume  of  the  pepsin  solution  originally  taken  (20  c.c.). 
With  this  solution  repeat  (a)  and  (b). 

5.  Trypsin.-j- — Free  a  beef  pancreas  from  fat,  cut  up  fine,  and 
weigh ;  transfer  to  wide-mouth  bottle  and  add  3  c.c.  10  per  cent, 
alcohol  for  each  gram  of  pancreas.     Add  5  c.c.  of  chloroform, 
cork  tightly,  shake,  and  set  aside  for  two  or  three  days. 

Then  take  out  25-35  c.c.  of  the  clear  liquid  and  pour  on  a 
filter.  Stopper  tightly  again,  and  put  the  bottle  in  the  warm  room 
(or  incubator)  to  be  preserved  for  later  experiments  on  "amino 
acids."  With  the  filtered  portion  of  the  pancreatic  extract  make 
the  two  following  experiments : 

(a)  Test  its  digestive  power  on  egg  albumin  (Mett's  tubes), 

(b)  Determine  the  effect  of  heat  (75°  for  15  minutes)  en  the 

digestive  power  of  the  trypsin  solution. 

6.  Malt  Diastase. — Heat  10  g.  of  dry  malt  in  a  dry  test  tube 
to  100°  for  15  minutes  (in  boiling  water  bath).     The  test  tube 

*  See  Hammarsten :    "Pepsin" — "Pepsin  Test." 
|  See  Hammarsten :     "Trypsin." 

27 


must  be  submerged  to  as  great  a  depth  as  the  height  of  the  malt 
within  the  tube.  Digest  with  100  c.c.  water  over  night  at  room 
temperature.  Filter. 

Digest  10  g.  of  unheated  malt  with  100  c.c.  water  over  night 
as  before.  Filter. 

With  the  malt  filtrates  and  a  thin  boiled  starch  paste  ( I  g.  starch 
to  100  c.c.  water)  make  the  following  experiments: 

Mix  5  c.c.  of  the  starch  paste  with  15  c.c.  of  each  malt  extract, 
and  let  stand  at  75-80°  (in  a  beaker  of  water). 

In  each  case  determine  approximately  when  all  the  starch  has 
disappeared.  To  do  this,  transfer  at  five-minute  intervals  a  few 
drops  of  the  digestive  fluid,  by  means  of  a  glass  tube,  from  the 
test  tube  into  a  porcelain  evaporating  dish,  and  add  drop  by  drop 
dilute  iodin  solution  (lodin  Test). 

Heat  5  c.c.  of  a  diastase  solution  to  boiling,  cool,  and  test 
(at  75-80°  C.)  its  power  to  digest  starch. 

7.  Reversible  Reactions  (Mass  Law).* — Mix  5  c.c.  methyl  ace- 
tate, loo  c.c.  water,  and  I  drop  concentrated  sulphuric  acid  in  a 
small  flask  (200-300  c.c.). 

(a)  Titrate  5  c.c. 

(b)  Boil  for  about  5  minutes,  using  reflux  condenser.     Cool, 
remove  5  c.c.,  titrate  the  acidity  (what  indicator?),  and  calculate 
the  acidity  for  100  c.c. 

Continue  the  boiling  for  one  hour,  and  repeat  the  titration. 

(c)  Mix  5  c.c.  of  glacial  acetic  acid  with  100  c.c.  of  methyl 
alcohol,  and  add  one  drop  of  concentrated  sulphuric  acid.     Re- 
move 5  c.c.,  dilute  this  with  water,  and  titrate  the  acidity.    Intro- 
duce the  preparation  into  a  250  c.c.  flask  attached  to  a  reflux 
condenser,  and  boil  for  two  hours.     Cool,  remove  5  c.c.,  dilute, 
and  titrate  as  before. 

*  See  Hammarsten. 


PART  III 
FATS 

1.  Solubility  of  Fats.— Test  the  solubility  of  tallow  in  water, 
5  per  cent.  NaOH,  ether,  chloroform,  and  alcohol,  carefully  avoid- 
ing the  vicinity  of  a  flame. 

Let  a  drop  of  the  ether  solution  fall  on  paper,  and  note  the 
result. 

Dissolve  in  3  c.c.  of  warm  benzol  enough  tallow  to  give  a 
moderate  precipitate  on  cooling.  Place  some  of  the  precipitate  on 
a  slide  under  a  cover  glass,  examine,  and  describe.  Note  espe- 
cially the  shape  of  the  ends  of  the  individual  crystals. 

2.  "lodin  Number."    Degree  of  TJnsaturation  of  Fats;  WijY 
Method. — Start   simultaneously  the  determination  of  the  iodin 
number  of  cottonseed  oil  and  beef  tallow.    Weigh  about  .3  g.  of 
cottonseed  oil,  or  about  i  g.  of  beef  tallow,  into  a  250  c.c.  flask, 
and  dissolve  in  chloroform  (10  c.c.).     Add  25  c.c.  Wijs'  iodin 
solution  with  a  pipet,  stopper,  and  put  in  a  dark  place  for  half 
an  hour.    Add  15  c.c.  of  10  per  cent,  potassium  iodid,  and  dilute 
with  100  c.c.  of  water,  titrate  the  excess  of  iodin  (partly  in  solu- 
tion in  the  water,  partly  in  the  chloroform)  with  .1  N  sodium 
thiosulphate,  by  running  the  latter  into  the  flask  until,  after  re- 
peated shaking,  both  the  chloroform  and  the  watery  solution  are 
but  faintly  straw  colored.    Then  add  a  few  drops  of  a  I  per  cent, 
starch  solution,  and  continue  the  titration  to  the  disappearance  of 
the  blue  color. 

While  waiting  for  absorption  to  take  place,  the  value  of  the 
iodin  solution  may  be  determined  in  terms  of  .1  N  thiosulphate 
by  adding  KI  and  titrating  in  the  same  way  as  above.  The 
difference -bet  ween  the  two  values  represents  the  amount  of  iodin 
absorbed  by  the  fat,  and  is  calculated  in  grams  of  iodin  per  100  g. 
of  fat. 


Example : 

.3  g.  cottonseed  oil,  when  treated  as  above,  required  35  c.c.  of 
.1  N  thiosulphate  for  back  titration. 

25  c.c.  of  the  iodin  solution  required  60  c.c.  of  .1  N  thiosulphate. 

The  oil  therefore  absorbed  iodin  corresponding  to  60  —  35  = 
25  c.c.  .1  N  thiosulphate,  i.e.,  25  c.c.  .1  N  iodin  or  25  X  .0127  g. 
Iodin  —  .317  g.  I.  The  Iodin  Number  is,  therefore,  AJ-ft  X  .31?  = 
105.6. 

Wijs'  IODIN  SOLUTION. — Dissolve  13  g.  of  iodin  in  I  liter  of 
glacial  acetic  acid.  Titrate  the  iodin  content  of  the  solution,  and 
then  pass  washed  and  dried  chlorin  gas  into  the  solution  until  the 
titration  number  is  doubled.  A  very  distinct  change  in  the  color 
of  the  solution  indicates  when  this  has  taken  place. 

The  thiosulphate  solution  is  prepared  by  dissolving  24  g.  of  the 
crystallized  salt  in  I  liter  of  water  and  standardizing  it  in  the  usual 
way  (see  page  127). 

3.  Saponification  and  Preparation  of  Fatty  Acids. — Boil  about 
20  g.  of  beef  tallow  with  100  c.c.  of  alcoholic  solution  of  sodium 
hydrate  on  a  water  bath  over  night,  or  until  the  residue  is  dry. 
To  the  mixture  add  about  300  c.c.  water  and  heat  to  boiling. 
To  the  hot  solution  add  a  few  drops  of  methyl  orange;  while 
continuing  the  heating   (and  stirring),  acidify  with  dilute  sul- 
phuric acid,  and  filter.    Save  the  filtrate  which  contains  glycerin, 
then  wash  the  fatty  acid  residue  several  times  with  hot  water. 
Throw  away  the  washings. 

Transfer  the  "glycerin  filtrate"  to  an  evaporating  dish,  label, 
and  place  on  the  water  bath  for  evaporation  to  dryness. 

4.  Solubility  of  Fatty  Acids.— Test  the  solubility  of  the  fatty 
acid  mixture  prepared  from  tallow  in  water,  5  per  cent.  NaOH, 
ether,  alcohol,  and  benzol.     Compare  the  results  with  those  ob- 
tained with  fat. 

Let  a  drop  of  the  ether  solution  fall  on  paper,  and  note  the 
result. 

Dissolve  enough  of  the  fatty  acid  mixture  in  warm,  alcohol  to 
give  moderate  precipitate  on  cooling.  Examine  the  crystals  under 
the  microscope,  and  describe  as  in  the  case  of  the  fat  crystals. 

5.  Spontaneous  Saponification  of  Fats. — Dissolve  about  .5  g. 
tallow,  or  a  few  drops  of  oil,  in  10  c.c.  warm  alcohol  in  a  test 

33 


tube  (avoid  fire!).  Add  3-4  drops  phenolphthalein  solution,  and 
then,  drop  by  drop,  tenth  normal  sodic  hydrate  solution  (alcoholic 
sodic  hydrate  solution  is  best)  until  the  indicator  reveals  a  dis- 
tinctly alkaline  reaction.  Let  the  mixture  stand  in  a  warm  room 
over  night,  and  again  add  alkali  (drop  by  drop)  until  the  alkaline 
reaction  reappears. 

6.  Titration  of  Higher  Fatty   Acids. — Dissolve  about   .2  g. 
fatty  acid  mixture  in  10  c.c.  warm  alcohol  or  benzol  (avoid  fire  !). 
Add  3-4  drops  phenolphthalein,  and  titrate  with  tenth  normal 
alcoholic  sodic  hydrate  solution  until  an  alkaline  reaction  is  ob- 
tained.    One  cubic  centimeter  of  the  alkali  corresponds  to  how 
much  fatty  acid  ? 

On  the  basis  of  the  solubilities  and  reactions  of  fats,  fatty 
acids,  and  soaps,  work  out  a  scheme  for  their  separation  and 
identification.  Apply  the  scheme  to  three  unknowns  furnished. 

7.  Fat  Digestion  with  Lipase    (Castor  Bean). — Remove  the 
shells  from  10  g.  fresh  castor  beans,  break  -them  up  as  fine  as 
possible,  and  allow  to  stand  over  night  in  a  loosely  stoppered 
test  tube  full  of  alcohol  ether  mixture.    Pour  off,  grind  the  beans 
to  a  powder  in  a  small  mortar,  transfer  to  a  test  tube,  and  let 
stand  under  ether  over  night.    Filter  with  suction,  and  wash  two 
or  three  times  with  small  amounts  of  the  alcohol  ether  mixture. 
Grind  with  the  powder  in  the  order  named,  5  c.c.  .1  N  sulphuric 
acid   (supplied),  5  c.c.  of  neutral  cotton  oil   (Sp.  gr.  .92),  and 
5  c.c.  lukewarm  water.    The  water  should  be  added  a  little  at  a 
time  and  thoroughly  worked  into  the  mixture  so  that  at  the  end 
of  the  operation  a  good  emulsion  is  secured.     Cover  the  evapo- 
rating dish,  and  let  stand  in  a  warm  place  over  night. 

Add  50  c.c.  of  alcohol,  10  c.c.  of  ether,  and  a  few  drops  of 
phenolphthalein,  and  titrate  with  .5  N  sodium  hydrate.  Calculate 
the  amount  of  fatty  acid  and  the  per  cent,  of  fat  digested. 

8.  Glycerin  and  the    Acrolein  Test. — To  about  5  g.  acid  po- 
tassium sulphate  (KHSO4)  in  a  porcelain  crucible  add  one  drop 
of  glycerin,  heat  over  a  direct  flame,  and  note  the  pungent  odor 
and  tear-begetting  quality  of  the  fumes.     Note  how  much  heat 
must  be  applied  to  secure  an  unmistakable  test. 

When  the  filtrate  (saved  from  the  saponification  of  beef  tallow) 
has  evaporated  to  dryness,  the  residue  obtained  is  sodium  sul- 

35 


phate  ;  mixed  with  it  there  should  be  some  glycerin.     ( How  much 
glycerin  might  be  there?) 

Mix  with  a  glass  rod  2-3  g.  of  this  residue  with  5-6  drops 
concentrated  sulphuric  acid  in  a  dry  crucible,  and  apply  heat.  If 
an  unmistakable  acrolein  test  is  not  obtained,  repeat  with  more 
of  the  residue. 

9.  Emulsification. — Put  1-2  c.c.  of  a  solution  of  sodium  car- 
bonate (.2  per  cent.)  in  a  watch  glass,  and  place  in  the  center  a 
drop  of  rancid  oil.    The  oil  soon  shows  a  white  rim,  and  a  milky 
opacity  spreads  over  the  solution.    Note  with  the  microscope  the 
active  movements  in  the  vicinity  of  the  fat  drop,  due  to  the  sepa- 
ration of  minute  particles  of  oil. 

Examine  a  sample  of  milk  under  the  microscope.  The  fat 
should  be  in  a  state  ©f  fine  emulsion. 

10.  Lecithin. — Demonstrate  myelin  movements  (observed  on 
mixing  lecithin  with  water). 

Mix  a  small  piece  of  lecithin  in  water  in  a  test  tube.  Shake 
vigorously  for  a  time,  and  state  what  occurs.  To  the  contents 
add  concentrated  caustic  soda  and  boil.  Note  the  fishy  odor  of 
trimethylamine  (from  the  cholin).  Acidify  the  solution — fatty 
acids  are  precipitated,  and  glycerophosphoric  acid  is  left  in  solu- 
tion. Write  the  graphic  formula  for  lecithin. 

11.  Cholesterin;  Liebermann's  Reaction.-^Dissolve  a  crystal  of 
cholesterin  in  10  c.c.  of  dry  chloroform,  and  to  this  solution  add 
a   few  drops  of  acetic  anhydrid    (formula?)    and  one  drop  of 
concentrated  H2SO4.    Shake.    The  liquid  becomes  rose  red,  blue, 
then  dark  green. 


37 


PART  IV 
CARBOHYDRATES 

One  of  the  marked  characteristic  properties  of  the  monosac- 
charids  is  the  ease  with  which  under  certain  conditions  they  ab- 
stract oxygen  from  the  hydroxids  of  the  heavy  metals,  setting 
free  either  the  metals  or  their  lower  oxids.  Most  of  the  reduction 
tests  for  "sugar"  are  based  on  this  fact.  Because  the  occurrence 
of  sugar  in  urine  is  of  clinical  importance  and  because  it  is  some- 
times difficult  to  determine  with  certainty  whether  a  given  urine 
does  or  does  not  contain  abnormal  amounts  of  sugar,  a  great 
many  reduction  tests,  with  several  modifications  of  each,  have 
been  proposed — all  designed  to  facilitate  the  detection  of  abnormal 
amounts  of  sugar  in  human  urine. 

1.  Trommer's  Test  for  Sugar. — Trommer's  test  represents  the 
oldest  and  simplest  of  the  many  methods  of  testing  for  sugar  by 
means  of  its  ability  to  reduce  cupric  hydroxid  to  (greenish  yellow, 
yellow,  or  reddish)  cuprous  oxid. 

To  about  5  c.c.  of  one  per  cent,  dextrose  solution  in  a  test  tube 
add  3  c.c.  ten  per  cent,  sodic  hydrate  solution  (or  I  c.c.  of  a 
saturated  solution),  and  then  add  drop  by  drop^  with  shaking, 
one  per  cent,  copper  sulphate  solution  (or  saturated  copper  ace- 
tate solution)  so  long  as  the  copper  hydroxid  formed  readily 
dissolves  on  shaking.  Heat  the  solution,  or  the  upper  part  of  it, 
nearly  to  boiling,  and  note  the  character  of  the  ensuing 
reduction. 

In  the  case  of  urines  with  traces  of  sugar,  Trommer's  test  may 
fail  because  too  much  copper  (yielding  black  copper  oxid)  or  too 
little  (yielding  no  precipitate)  was  used.  To  obviate  these  dif- 
ficulties there  have  been  proposed  many  different  cupric  hydroxid 
reagents  ia  which  the  surplus  cupric  hydroxid  is  kept  in  solution 
instead  of  being  precipitated. 

39 


2.  Fehling's  Test. — Fehling's  solution,  which  in  the  past  has 
been  extensively  used  for  the  quantitative  determination  of  sugar, 
is  made  up  as  follows : 

SOLUTION  i.  Copper  sulphate  solution.  Weigh  out  accurately 
17.325  g.  purest  obtainable  copper  sulphate.  Dissolve  in  hot 
water,  cool,  and  make  up  to  a  volume  of  500  c.c. 

SOLUTION  2.  Alkaline  tartrate  solution.  Weigh  (on  rough 
scales)  63  g.  potassium  hydroxid  and  100  g.  Rochelle  salt  (sodium 
potassium  tartrate),  transfer  to  a  beaker,  add  water,  stir  until 
dissolved,  and  dilute  to  a  volume  of  500  c.c. 

The  two  Fehling's  solutions  must  be  kept  in  separate  and  well- 
stoppered  bottles  (particularly  the  alkaline  tartrate  solution). 

When  used  for  quantitative  sugar  determinations,  the  copper 
sulphate  solution  must  be  measured  out  with  pipets  as  carefully 
as  any  other  standard  solution,  but  the  alkaline  tartrate  solution 
can  be  measured  out  with  cylinders.  Each  c.c.  of  the  copper  solu- 
tion corresponds  to  5  mg.  of  dextrose,  6.7  mg.  of  lactose,  7.4  mg. 
of  maltose,  or  4.75  mg.  of  "invert  sugar,"  when  completely  re- 
duced. 

The  two  component  parts  of  Fehling's  solution  are  kept  in 
separate  bottles,  because  when  mixed  the  solution  gradually  de- 
teriorates. For  qualitative  tests  it  is,  however,  perfectly  safe 
and  more  convenient  to  mix  enough  of  the  solutions  for  a  day's 
tests.  To  10  or  20  c.c.  of  each  solution  mixed  in  a  flask  add 
two  volumes  (40  or  80  c.c.)  of  water  and  shake.  With  this 
diluted  Fehling's  solution  the  test  for  sugar  is  made  as  follows : 

Pour  5-10  c.c.  of  Fehling's  solution  into  a  test  tube  and  heat 
to  boiling  (the  solution  must  remain  clear).  Now  add  one  or 
two  c.c.  of  sugar  solution,  or  of  urine,  and  again  heat  to  boiling. 
If  more  than  minute  traces  of  sugar  are  present,  cuprous  oxid  is 
precipitated.  The  mere  fading  of  the  solution  does  not  indicate 
sugar  in  urine. 

3.  Benedict's  Test. — One  of  the  best  qualitative  tests  for  sugar 
in  urine  by  means  of  copper  solutions  is  the  one  recently  pro- 
posed by  S.  R.  Benedict.     Benedict's  reagent  is  so  adjusted  that 
it  is  rather  more  sensitive  to  dextrose  than  Fehling's  solution, 
yet  is  not  reduced  by  creatinin  or  uric  acid,  and  little,  if  at  all, 
by  chloroform  (which  is  often  added  as  a  preservative  to  urine). 
Unlike  Fehling's  reagent  it  consists  of  a  single  solution.     The 
reagent  is  made  as  follows : 


Dissolve  85  g.  sodium  citrate  and  50  g.  anhydrous  sodic  car- 
bonate in  400  c.c.  of  water.  Dissolve  8.5  g.  copper  sulphate  in 
50  c.c.  of  hot  water.  Pour  the  copper  sulphate  solution  slowly, 
and  with  stirring,  into  the  alkaline  citrate  solution.  Filter  if 
necessary.  Label  and  preserve.  \  , 

Heat  to  boiling  about  5  c.c.  of  Benedict's  reagent  in  a  test  tube 
together  with  a  pebble  or  two,  to  prevent  bumping.  Add  about 
i  c.c.  of  sugar  solution  (or  urine)  and  boil  for  1-2  minutes.  If 
more  than  two-  or  three-tenths  per  cent,  of  sugar  is  present,  the 
solution  will  be  filled  with  a  colloidal  (greenish,  yellow,  or  red- 
dish) precipitate.  With  smaller  amounts  of  sugar  the  precipitate 
will  usually  appear  only  on  cooling  (the  cooling  should  not  be 
hastened  by  immersion  in  cold  water). 

4.  Reduction  Test  for  Sugar  in  Normal  Urine.— That  most 
human  urines  contain  distinct  traces  of  reducing  sugar  can  be 
shown  by  the  use  of  more  sensitive  copper  reagents.  One  such 
reagent  can  be  made  as  follows : 

(A)  Dissolve  5  g.  of  crystallized  copper  sulphate  in  100  c.c. 
of  hot  water,  and  to  the  cooled  solution  add  60-70  c.c.  of  pure 
glycerin. 

(B)  Dissolve  125  g.  of  anhydrous  potassium  carbonate  (with 
stirring)  in  400  c.c.  water. 

Mix  one  volume  of  the  glycerin-copper  solution  (A)  with  two 
volumes  of  the  potassium  carbonate  solution  (B).  Only  small 
portions  should  be  mixed  at  a  time,  as  the  reagent  (after  mixing) 
does  not  keep,  but  undergoes  gradual  reduction. 

The  test  for  sugar  in  normal  urine  is  made  as  follows : 

First  transfer  5-10  c.c.  of  the  mixed  reagent  to  a  test  tube, 
and  boil  the  reagent  in  order  to  determine  the  extent,  if  any,  to 
which  the  reagent  is  spontaneously  reduced. 

Transfer  1-2  g.  picric  acid  to  a  small  bottle ;  to  it  add  some  of 
the  urine  (10-50  c.c.)  to  be  tested,  insert  a  cork,  shake  for  five 
minutes,  and  filter.  By  this  treatment  the  disturbing  creatinin 
and  uric  acid  are  removed.  Now  heat  5~IQ  c-c'  °^  the  copper 
solution  to  boiling  in  a  fairly  wide  test  tube  (with  a  pebble  to 
prevent  bumping),  add  1-2  c.c.  of  the  filtered  urine,  and  boil  for 
60-75  seconds.  If  the  sugar  present  is  relatively  large  in  amount, 
the  solution  becomes  turbid  while  still  boiling,  as  in  Benedict's 
test.  If  no  such  turbidity  is  observed,  centrifuge  at  once,  i.e., 
before  cooling.  The  bottom  of  the  centrifuge  will  be  covered 

43 


with  cuprous  oxid.     The  crystals  to  be  observed  in  the  super- 
natant cooling  liquid  are  potassium  picrate. 

Repeat  this  test  with  1-2  c.c.  urine  which  has  not  been  treated 
with  picric  acid,  and  note  how  much  more  abundant  the  reduction 
is  with  ordinary  creatinin-containing  urine. 

5.  Bismuth  Test, — Almen-Nylander's  test  for  sugar  depends 
on  the  ease  with  which  alkaline  solutions  of  bismuth  are  reduced. 
The  reagent  is  made  as  follows : 

To  4  g.  of  Rochelle  salt  in  a  small  flask  add  100  c.c.  10  per  cent, 
caustic  soda  solution  and  2  g.  of  bismuth  subnitrate.  Digest  on  a 
water  bath  for  an  hour.  Let  settle,  and  use  the  supernatant 
liquid. 

This  reagent  is  not  used  in  the  same,  way  as  the  copper  reagents. 
With  the  latter  a  little  of  the  urine  or  sugar  solution  is  added 
to  5-10  c.c.  of  the  reagent.  With  the  bismuth  solution  the  pro- 
portions of  reagent  and  urine  are  reversed. 

To  5-10  c.c.  of  sugar  solution  add  .5-1  c.c.  of  the  bismuth 
solution,  and  heat  (with  a  pebble)  for  2-3  minutes.  Set  aside 
to  cool  spontaneously.* 

The  bismuth  test  is  widely  used,  and  is  valuable  for  the  detec- 
tion of  abnormal  amounts  of  sugar  in  urine.  Albumin  interferes 
with  the  test,  and  when  present  must  be  removed.  Certain  drugs 
also  interfere  with  the  validity  of  the  test. 

6.  Relative  Sensitiveness  and  Significance  of  the  Reduction 
Tests  for  Sugar. — With  a  .2  per  cent,  solution  of  dextrose  deter- 
mine the  relative  sensitiveness  of  the  preceding  qualitative  tests 
for  sugar. 

Apply  the  sam~  tests  to  concentrated  normal  urine  (night 
urine).  Record  tne  results. 

7.  Phenylhydrazin  Test  (Osazone  Test). — To  5-10  c.c.  of  .2  per 
cent,  dextrose  solution  in  a  test  tube  add  5  c.c.  of  a  phenylhydrazin 
solution  (containing  5  per  cent,  phenylhydrazin  hydrochlorid,  20 
per  cent,  sodic  acetate,  and  10  per  cent,  acetic  acid).     Heat  in  a 
beaker  of  boiling  water  for  half  an  hour.     Let  the  test  tube 
remain  in  the  beaker  until  the  water  has  cooled,  and  examine  the 
glucose  osazone  crystals  under  the  microscope. 

Write  the  reaction  involved  in  the  formation  of  osazone. 

*  See  Hammarsten :     "Almen  Sugar  Test." 

45 


8.  Dextrose  Reactions  versus  other  Carbohydrates. — Apply  one 
of  the  copper  reducing  tests,  the  bismuth  test,  and  the  phenyl- 
hydrazin  test  to  .2  per  cent,  solutions  of  arabinose,  levulose,  cane 
sugar,  maltose,  and  lactose*. 

Apply  the  copper  reducing  test  also  to  .2  per  cent,  solutions  of 
gum  arabicum,  commercial  dextrin,  and  starch. 

In  the  case  of  any  of  the  above  carbohydrate  solutions  which 
have  yielded  negative  or  doubtful  results,  digest  20  c.c.  with  I  c.c. 
concentrated  hydrochloric  acid  in  boiling  water  for  half  an  hour 
(or  at  room  temperature  over  night),  and  repeat  the  test  (or 
tests)  made  on  the  original  solution.  Tabulate  and  explain  the 
results. 

9.  Selivanoff's  Test  for  Ketose-Sugars. — SelivanofFs   reagent 
contains  .05  per  cent,  of  resorcin  and  about  12  per  cent,  of  hydro- 
chloric acid. 

To  5-10  c.c.  of  the  reagent  in  a  test  tube  add  about  I  c.c.  of 
.1  per  cent,  solution  of  levulose,  boil  for  one  minute,  set  aside  to 
cool,  and  note  the  development  of  the  color. 

Repeat  the  test  with  dextrose  and  with  cane  sugar  (and,  if 
desirable,  with  other  dilute  sugar  or  carbohydrate  solutions). 
Tabulate  the  results. 

10.  Test  for  Ketose  Sugars    (Levulose?)    in  Urine. — Collect 
urine  for  one  hour  (preferably  just  before  the  noon  hour).    Then 
take  50  g.  of  cane  sugar  and  collect  urine   for  another  hour. 
Dilute  the  smaller  volume  of  urine  to  that  of  the  larger,  or  both 
to  a  convenient  small  volume.    To  10  c.c.  of  each  in  a  test  tube 
add  5  c.c.  of  10  per  cent,  lead  acetate  solution,  shake,  and  filter. 
Apply  Selivanoff's  levulose  test  to  about  2  c.c.  of  each  filtrate. 
Record  the  results  obtained. 

Minute  traces  of  levulose  in  human  urine  are  probably  not  so 
infrequent  an  occurrence  as  is  generally  assumed  to  be  the  case. 
The  color  obtained  with  urine  is,  however,  not  exactly  like  that 
given  by  aqueous  levulose  solutions.  If  a  distinct  "levulose"  out- 
put from  the  cane  sugar  is  obtained,  test  both  urines  for  reducing 
sugar,  and  note  whether  there  appears  to  be  an  increase  of  sugar 
in  the  urine  of  the  second  period. 

oil.     Orcin  Test  for  Pentoses. — The  pentose  reagent  is  made  by 
dissolving  I  g.  of  orcin  in  500  c.c.  30  per  cent,  hydrochloric  acid 

47 


(6:  i)  and  adding  i  c.c.  of  10  per  cent,  ferric  chlorid  solution. 

Heat  5  c.c.  of  the  orcin  solution  to  boiling  in  a  test  tube.  Re- 
move from  the  flame,  and  add  (immediately)  i  c.c.  i  per  cent, 
solution  of  arabinose. 

Repeat,  adding  i  c.c.  .2  per  cent,  arabinose  solution. 

Note  the  color  (violet,  blue,  red,  green)  and  the  formation  of  a 
precipitate. 

Repeat  with  i  per  cent,  arabinose  solution  previously  diluted 
(a)  with  4  volumes  of  urine  (b)  with  4  volumes  i  per  cent, 
glucose  solution. 

Repeat  (a)  with  urine  alone  (b)  with  i  per  cent,  glucose  solu- 
tion (c)  with  i  per  cent,  cane  sugar  solution. 

12.  Fermentation  Test  for  Sugar, — Certain  sugars  (which?) 
are  decomposed  by  yeast  into  carbon  dioxid  and  alcohol.  The 
formation  of  CO2  in  fermentation  has  been  extensively  used 
both  for  qualitative  studies  and  for  quantitative  determinations 
of  sugar.  Except  among  physicians  who  have  not  the  facilities 
for  making  other  tests,  the  fermentation  method  is  now  seldom 
used. 

To  some  .5  per  cent,  sugar  solution  (dextrose,  pentose,  cane 
sugar,  lactose)  in  a  test  tube  add  a  small  piece  of  yeast.  Shake 
to  make  uniform  mixtures,  and  with  each  fill  a  "fermentation 
tube."  Substitute  water  for  sugar  solution  in  a  control  test,  and 
set  the  tubes  aside  in  a  warm  place  over  night.  Record  the 
results. 

^13.  Benedict's  Method  for  the  Determination  of  Sugar. — Pre- 
pare 500  c.c.  of  Benedict's  solution  as  follows :  Dissolve  9  g. 
pure  copper  sulphate  in  a  500  c.c.  volumetric  flask  with  about 
100  c.c.  distilled  water.  Dissolve  50  g.  anhydrous  sodic  carbonate, 
100  g.  sodium  citrate,  and  65  g.  potassium  sulphocyanate,  in 
250  c.c.  distilled  water.  The  copper  sulphate  must  be  weighed 
accurately  on  the  analytical  balance.  Pour  the  copper  solution, 
slowly,  with  stirring  and  without  loss  of  a  single  drop,  into  the 
alkaline  citrate  solution.  Then  pour  the  mixed  solution  back  into 
the  measuring  flask  without  loss,  add  5  c.c.  5  per  cent,  potassium 
f  errocyanide  solution,  and  with  the  rinsings  make  the  total  volume 
up  to  500  c.c.  Mix,  transfer  to  a  clean,  dry  bottle,  label,  and 
preserve.  Twenty-five  cubic  centimeters  of  the  solution  corre- 
sponds to  50  mg.  of  dextrose,  52  mg.  of  levulose,  67  of  lactose, 
or  74  of  maltose. 

49 


The  determination  is  carried  out  as  follows: 

Measure  25  c.c.  of  Benedict's  solution  into  a  porcelain  dish,  add 
5-10  g.  of  solid  sodic  carbonate,  heat  to  boiling,  and  while  boiling 
run  in  the  sugar  solution  (or  urine)  fairly  rapidly  until  a  white 
precipitate  begins  to  form.  Then  add  the  solution  more  slowly 
(with  slower  boiling)  until  the  last  trace  of  the  blue  color  dis- 
appears. The  addition  of  the  sugar  solution  should  be  done  at 
such  a  speed  that  the  boiling  solution  is  kept  nearly  constant  in 
volume  during  the  operation.  The  original  sugar  solution  (or 
urine),  if  concentrated,  should  be  diluted  so  that  not  less  than 
10  c.c.  will  be  required  to  give  the  amount  of  sugar  which  the 
25  c.c.  of  reagent  is  capable  of  oxidizing. 

Five  divided  by  the  volume  of  sugar  solution  taken  gives  the 
per  cent,  of  sugar.  Check  the  value  of  the  reagent  by  determin- 
ing the  sugar  in  .5  per  cent,  dextrose  solution. 

fj  14.  Polar iscope  Method  for  the  Determination  of  Sugar. — The 
specific  rotation  of  a  substance  is  the  angle  through  which  the 
plane  of  polarized  light  is  turned  by  I  dm.  of  a  solution  containing 
i  g.  of  the  optically  active  substance  per  c.c. 

A  definite  temperature  and  light  of  a  definite  wave  length 
(sodium  light)  must  be  used  in  determining  specific  rotations. 

The  angle  of  rotation  is  determined  by  means  of  some  form 
of  "polariscope"  (polarimeter,  saccharimeter,  etc.)  ;  and  the  spe- 
cific rotation  is  calculated  according  to  the  following  formula: 

c     .,-  Observed  Rotation  X  100 

Specific 

Rotation       Percentage  X  Length  of  Observation  Tube  (dm.) 

If  the  specific  rotation  is  known,  as  in  the  case  of  the  common 
sugars,  the  per  cent,  of  sugar  is  obtained  by  the  following  trans- 
position of  the  above  formula: 

_  Observed  Rotation  X  100 

~  Specific  Rotation  X  Length  of  Tube  (dm.) 

Following  are  the  specific  rotations  (yellow  light)  of  some  com- 
mon sugars : 

Glucose,  52.8;  Fructose,  —90;  Cane  Sugar,  66.5;  "Invert 
Sugar,"  — 20;  Lactose,  52.4;  Maltose,  137. 

51 


The  determination  of  sugar  by  means  of  the  polariscope  is  as 
follows : 

Rinse  the  polariscope  tube  (length  usually  I  or  2  dm.)  with 
the  sugar  solution,  and  fill  almost  to  overflowing.  Place  the  glass 
plate  over  the  open  end  in  such  a  way  that  the  .tube  does  not 
contain  any  air  bubble,  and  screw  on  the  cap.  Place  the  tube 
in  the  groove  of  the  polariscope.  Light  the  lamp,  and  move  the 
eyepiece  back  and  forth  until  the  lines  which  divide  the  field  are 
seen  sharp,  without  effort.  Then  turn  the  screw  until  the  several 
divisions  of  the  field  are  equally  illuminated,  and  take  a  reading  by 
means  of  the  vernier.  The  circle  upon  the  disk  of  the  apparatus 
is  divided  into  quarter  degrees ;  24  divisions  of  the  vernier  cor- 
respond in  length  to  .25°.  Consequently  every  division  of  the 
vernier  corresponds  to  .01°.  Ascertain  whether  the  disk,  start- 
ing from  its  middle  point,  has  been  moved  to  the  right  or  to  the 
left  of  the  zero  point  of  the  vernier.  Read  off  the  number  of 
whole  degrees  and  hundredths.  Take  several  readings  by  mov- 
ing the  lever  and  coming  back  again  to  the  point  where  the  dif- 
ferent parts  of  the  field  are  equal.  Correct  for  the  zero  point 
of  the  instrument  by  taking  readings  with  the  tube  filled  with 
water.  This  value  is  added  to,  or  subtracted  from,  the  reading 
found  with  the  sugar  solution. 

If  a  saccharimeter  is  used  instead  of  the  general  circular  polari- 
scope, the  reading  on  the  scale  is  converted  into  angular  degrees 
of  rotation  by  multiplying  by  the  factor  .345.  The  percentage  of 
sugar  in  the  solution  is  then  calculated  by  using  the  formula 
given  above. 

Prepare  100  c.c.  dextrose  solution  containing  about  5  per  cent, 
of  sugar.  The  amount  of  sugar  taken  should  be  weighed  accu- 
rately. To  overcome  the  "muta  rotation"  of  the  dextrose  solu- 
tion either  heat  the  solution  to  100°  and  cool,  or  allow  to  stand 
over  night  (or  add  first  one  drop  of  ammonia  and  then  one  drop 
of  acid). 

Determine  the  sugar  content  of  the  solution  by  the  polari- 
scope, and  also  (after  appropriate  dilution)  by  titration  with 
Benedict's  copper  solution. 

Determine  by  titration,  and  also  by  the  optical  rotation,  the 
dextrose  content  of  three  unknown  "sugar  urines." 

Before  the  sugar  in  urine  can  be  determined  by  the  polari- 
scope it  is  often  necessary  to  "clear"  it  by  the  addition  of  a 
pinch  of  basic  lead  acetate  and  filtration. 

53 


15.  Cane  Sugar. — Prepare  100  c.c.  of  a  5  per  cent,  cane  sugar 
solution  by  the  help   of  the  balance.     Determine  the  concen- 
tration of  the  solution  with  the  polariscope. 

Transform  a  part  of  the  solution  into  "invert  sugar,"  and  de- 
termine the  sugar  content  by  titration  with  Benedict's  solution. 

The  inversion  is  carried  on  as  follows : 

Transfer  to  a  100  c.c.  volumetric  flask  10  c.c.  of  the  cane  sugar 
solution,  and  add  5  c.c.  of  water  and  I  c.c.  concentrated  hydro- 
chloric acid.  Heat  water  in  a  large  beaker  or  porcelain  dish  to 
70°  C.  When  the  water  bath  has  reached  70°  C,  immerse  the 
flask  in  the  water,  rotate  it  continuously  for  ten  minutes,  and  at 
once  dilute  to  the  100  c.c.  mark  with  cold  water.  Mix,  and  titrate 
the  sugar  content  as  in  the  case  of  dextrose,  giving  25  c.c.  of  the 
copper  solution  a  value  of  51  instead  of  50  mg.  of  sugar  (why?). 

16.  Problem. — On  the  basis  of  the  tests  and  determinations 
made  with  dextrose,  levulose,  pentose,  and  cane  sugar,  work  out 
a  scheme  for  identifying  (a)  dextrose  and  levulose  (b)  dextrose 
and  pentose  (c)  dextrose  and  cane  sugar — when  any  one  of  these 
pairs  is  present.    Apply  to  the  unknowns  furnished. 

17.  Preparation  of  Maltose. — Mix  10  g.  of  starch  with  30  c.c. 
of  cold  water  until  a  smooth  paste  is  obtained.    Pour  this  slowly, 
and  with  stirring,  into  250  c.c.  of  boiling  water  in  a  large  beaker, 
continue  the  boiling  1-2  minutes,  let  cool  to  75°  C.,  stir  in  I  tea- 
spoonful  of  malt,  and  keep  at  this  temperature  until  the  mixture 
becomes  thin  and  watery.     Heat  again  to  boiling  with  stirring, 
cool  to  75°,  add  another  teaspoonful  of  ferment,  and  keep  at 
this  temperature  until  a  sample  no  longer  gives  any  color  with 
iodin  solution   (about  two  hours).     Boil,  filter  (taste),  and  de- 
termine the  sugar  quantitatively  in  a  sample  of  the  filtrate  (a)  as 
it  stands,  (b)  after  heating  on  the  water  bath  with  hydrochloric 
acid  for  about  two  hours.     Explain. 

18.  Preparation  of  Milk  Sugar. — To  300  c.c.  of  skimmed  milk 
diluted  with  800  c.c.  of  water,  add  cautiously  2  per  cent,  acetic 
acid  to  precipitate  the  casein.    When  enough  has  been  added  the 
liquid  is  nearly  clear.    Filter.     Boil  the  filtrate,  and  filter  off  the 
coagulated  albumin.     Evaporate  the  filtrate  on  the  water-bath 
to  a  thin  syrup,  and  allow  it  to  stand  until  the  sugar  has  crystal- 
'ized  out. 

55 


19.  Starch. — Rub  2  g.  of  starch  to  a  thin  paste  with  a  little 
water,  and  pour  the  mixture  slowly,  with  constant  stirring,  into 
300  c.c.  of  boiling  water. 

To  5  c.c.  of  the  starch  paste  and  10  c.c.  water  in  a  test  tube 
add  a  drop  of  iodin  solution.  Note  the  result.  Heat  the  solution 
gradually.  What  change  takes  place?  Cool  and  observe  the 
result.  Then  boil  and  again  allow  to  cool. 

Add  a  few  drops  of  a  solution  of  tannic  acid  to  a  few  cubic 
centimeters  of  the  dilute  starch  solution.  Warm. 

Test  a  portion  of  the  starch  solution  for  reduction. 

To  25  c.c.  of  saliva  in  a  small  flask  add  25  c.c.  of  the  starch 
paste.  Mix,  and  test  a  small  portion  for  sugar.  Digest  the 
remainder  at  40°  C,  and  test  for  sugar  and  for  starch  at  the 
end  of  15  minutes,  30  minutes,  and  two  hours. 

Mix  a  little  uncooked  starch  with  saliva,  expose  to  a  tempera- 
ture of  40°  C.  for  fifteen  minutes,  and  test  for  sugar. 

2(X  Dextrin. — Dissolve  a  little  commercial  dextrin  *  in  water, 
and  make  the  reduction  test  and  the  test  for  starch. 

Saturate  5  c.c.  of  the  dextrin  solution  with  solid  ammonium 
sulphate.  Filter.  Test  the  filtrate  with  iodin.  Remove  precipi- 
tate from  filter,  dissolve  in  a  little  hot  water,  cool,  and  test  with 
iodin. 

Boil  5  c.c.  of  the  solution  with  an  equal  volume  of  dilute  (10 
per  cent.)  hydrochloric  acid  for  5  minutes.  Test  for  sugar.  Save 
the  remainder  of  the  dextrin  solution  for  comparison  with  gly- 
cogen. 

Test  Graham  cracker  or  toast  for  dextrin  by  extracting  with 
cold  water,  filtering,  and  applying  the  iodin  test. 

21.  Glycogen. — Cut  four  fresh  (Why  fresh?)  oysters  into 
small  pieces,  and  throw  into  four  times  their  weight  of  boiling 
water  slightly  acidulated  with  acetic  acid.  After  boiling  for  a 
short  time,  remove  the  pieces,  grind  in  a  mortar  with  some  sand, 
return  to  the  water,  and  continue  the  boiling  for  several  minutes. 
Filter  while  hot.  The  opalescent  solution  thus  obtained  is  an 
aqueous  solution  of  glycogen  and  other  substances. 

*  Commercial  dextrin  sometimes  contains  unaltered  starch,  and  usually 
contains  reducing  sugar;  in  the  former  case  iodin  may  give  a  blue  or 
violet  (mixture  of  red  and  blue)  color;  in  the  latter  a  reduction  takes 
place  in  Fehling's  test.  Dextrin  gives  no  reaction  with  Fehling's  solution. 

57 


With  the  solution  of  glycogen  thus  obtained,  make  the  follow- 
ing tests : 

Add  iodin  solution  drop  by  drop  to  a  portion  of  the  glycogen 
solution.  The  liquid  will  assume  a  dark  red  color.  This  color 
disappears,  with  the  exception  of  the  color  due  to  the  iodin,  upon 
gentle  heating,  and  reappears  upon  cooling.  (Compare  with 
dextrin.) 

Test  the  glycogen  solution  with  Benedict's  solution  and  note 
the  result. 

Add  some  saliva  to  a  portion  of  the  glycogen  solution,  and 
put  in  the  warm  room  until  the  next  day.  Remove  and  divide 
into  two  portions.  Test  one  with  iodin  solution  for  glycogen, 
the  other  for  sugar.  Report  the  result. 


59 


PART  V 

PROTEINS 

A  fundamentally  important  general  consideration  to  be  noted  in 
the  study  of  protein  materials  is  that  proteins  are  colloids,  and  that 
colloidal  solutions  differ  materially  in  their  physical  and  chemical 
reactions  from  the  corresponding  reactions  of  ordinary  true  solu- 
tions (of  crystalloids).* 

1.  Dialysis  of  Colloidal  and  Molecular  Solutions.— In   a   parch- 
ment  dialyzing  tube   place    15    c.c.   of   blood   serum    (protein)    and 
i   c.c.  of  strong  salt  solution,   Na2SO4.     The  protein   is   present  in 
colloidal  form,  the  salts  in  molecular  (ionic)   solution.     Suspend  the 
tube  in  a  500  c.c.  beaker  of  distilled  water,  and  let  the  whole  stand 
over  night.     Test  a  portion  of  the  dialysate  for  protein  by  boiling 
in  a  test  tube  with   one  drop   of   dilute   acetic   acid.     Has   protein 
passed  through  the  membrane?    Test  the  dialysate  for  chlorids  and 
sulphates.     Do  salts  dialyze  through  parchment  membranes? 

2.  Suspension  Colloids;  Suspensoids.— PREPARATION. — Prepare   a 
colloidal   solution  of  gum  mastic  as   follows:     Drop   from   a  buret 
i  c.c.  of  saturated  alcoholic  solution  of  gum  mastic,  slowly  and  with 
stirring,  into  100  c.c.  of  distilled  water.    Filter. 

ACTION  OF  ELECTROLYTES. — To  10  c.c.  portions  of  the  above  filtrate 
add 

1.  5  c.c.  tenth  normal  HC1, 

2.  5  c.c.  tenth  normal  NaOH, 

3.  5  c.c.  normal  NaCl, 

4.  5  c.c.  urea  solution, 

5.  5  c.c.  concentrated  cane  sugar  solution. 
Let  stand,  note  what  occurs,  and  explain. 

EFFECT  OF  HEAT. — Boil  a  portion  of  the  solution  in  a  test  tube. 

*  Fats  and  the  more  complex  carbohydrates  behave  also  as  colloids ; 
so  indeed  do  all  substances  which  are  insoluble  in  water,  when  suspended 
in  water  in  a  state  of  sufficiently  fine  division.  Colloidal  solutions  of 
metallic  platinum  (one  of  the  least  soluble  of  metals)  are  frequently 
employed  in  the  study  of  certain  ferment  reactions.  Proteins  occur  in 
nature  almost  wholly  in  the  form  of  colloidal  solutions. 

61 


Evaporate  10  c.c.  of  solution  to  dryness  on  the  water  bath,  and 
try  to  redissolve  it. 

3.  Emulsion  Colloids;  Emulsoids.— PREPARATION. — To  5  g.   gel- 
atin in  a  beaker  add  60  c.c.  of  water,  and  heat  gently  with  constant 
stirring  until  the  gelatin  is  all  dissolved. 

ACTION  OF  ELECTROLYTES. — To  10  c.c.  portions  of  the  gelatin 
solution  add, 

1.  A  few  drops  10  per  cent.  NaCl, 

2.  A  few  drops  concentrated  HC1. 

Evaporate  10  c.c.  to  dryness  on  the  water  bath,  and  try  to  redis- 
solve. 

REVERSIBILITY  OF  COAGULATION  BY  ELECTROLYTES. — To  25  c.c.  of  a 
protein  solution  add  solid  ammonium  sulphate  with  shaking  until 
the  solution  is  saturated,  and  note  what  happens.  Filter  and  press 
the  precipitate  as  dry  as  possible  between  layers  of  filter  paper. 
Try  to  redissolve  the  precipitate  in  water. 

REVERSIBILITY  OF  COAGULATION  BY  HEAT. — To  another  25  c.c.  por- 
tion of  the  same  solution  add  2-3  drops  dilute  acetic  acid.  Heat  to 
boiling.  Filter,  press  the  precipitate  dry  as  before,  and  try  to  re- 
dissolve  in  water.  Tabulate  the  differences  between  emulsoids  and 
suspensoids. 

4.  "Hydrophile"  Colloids.* — To  some  gelatin  in  a  test  tube  add 
just  sufficient  water  to  cover  it.     Let  stand  and  note  changes. 

5.  Reversibility  of  "Sol"  and  "Gel"  States — Gelatinization  as 
a  Special  Case  of  Coagulation.  — Warm  the  above  mixture  until  so- 
lution is  complete.     Cool  under  the  cold  water  tap.     What  occurs? 
Repeat  the  heating  and  cooling  a  number  of  times. 

6.  Positively  and  Negatively  Charged  Colloids.— Place    a    few 
c.c.  of  colloidal  Fe(OH)3  solution  in  one  small  beaker,  and  a  few 
c.c.  of  colloidal  As2S3  solution   in  another.     Hang  a  long,  narrow 
strip  of  filter  paper  over  each  beaker  so  that  the  lower  end  touches 
the  liquid.    Let  stand  over  night.    Which  colloid  rises  to  the  greater 
height  ?     Explain. 

7.  Coagulation  by  Oppositively  Charged  Colloids,.— To    10    c.c. 
colloidal  arsenic  solution  add  10  c.c.  colloidal  Fe(OH)3.     Let  stand 
and  note  what  occurs. 

*  "Hydrophile"  colloids  are  one  class  of  emulsoids — not  all  emulsoids 
are  "hydrophilous." 


8.     Rate  of  Diffusion  of  Molecular  and  Colloidal  Solutions. -Melt 

some  4  per  cent,  gelatin  or  2  per  cent,  agar  jelly  in  a  beaker  by 
standing  in  hot  water.  Fill  5  small  test  tubes  1/3  full  and  allow 
to  solidify.  To  the  jelly  tubes,  1-5  respectively,  add  an  equal 
volume  of 

1.  Colloidal  As2S3, 

2.  Colloidal  Fe(OH)3, 

3.  Picric  acid, 

4.  Copper  sulphate, 

5.  Congo  red. 

Allow  to  stand  2  days.  Compare  the  speed  of  diffusion,  i.e.,  the 
distance  which  the  various  substances  have  penetrated  the  jelly. 
What  is  the  relation  between  the  size  of  particles  and  rate  of 
diffusion  ? 


1.  Test  for  Nitrogen,  Sulphur,  and  Phosphorus  in  Protein. — Put 

a  little  dry  protein  *  into  a  dry,  cheap  test  tube.  Add  a  piece  of 
metallic  sodium  the  size  of  a  pea,  and  heat  strongly  for  a  few 
minutes.  Cool.  Carefully  and  without  handling  the  material, 
break  into  a  dry  evaporating  dish.  Cover  the  substance  with  a 
wet  filter  paper.  After  five  minutes  cautiously  add  25  c.c.  water. 
Stir  well.  Filter  into  a  test  tube. 

(a)  To  5  c.c.  add  a  few  drops  of  ferrous  sulphate  and  a  drop 
of  ferric  chlorid  solution.    Warm  and  acidify  with  concentrated 
hydrochloric  acid,  noting  the  result. 

(b)  Acidify  5  c.c.  with  nitric  acid  and  add  a  few  drops  of 
ammonium  molybdate  solution.     Let  stand  over  night  and  look 
for  a  yellow  crystalline  precipitate. 

(c)  To  another  5  c.c.  add  a  few  drops  concentrated  sulphuric 
acid,  and  suspend  over  the  mouth  of  the  test  tube  a  piece  of  filter 
paper  previously  moistened  with  lead  acetate  solution. 

Discuss  the  action  of  the  sodium,  and  the  chemistry  of  the 
tests  (a),  (b),  (c). 

2.  Simple  Test  for  "Amid"  Nitrogen  and  for  Sulphur.— Put  a 
little  protein  in  a  test  tube  with  a  few  cubic  centimeters  of  strong 
sodium  hydrate  solution  and  three  drops  lead  acetate  solution. 
Heat  to  boiling,  and  suspend  a  piece  of  litmus  paper  over  the 
mouth  of  the  test  tube.    Explain. 

3.  Test  for  Phosphorus. — Boil  some  casein  in  the  hood  with 
10  c.c.   strong  nitric  acid   in   an   evaporating   dish.     Evaporate 
nearly  to  dryness,  add  25  c.c.  water,  and  test  for  phosphates. 

4.  Albumins. — Preparation  of  an  albumin  solution.    Consider- 
ing "egg  white"  to  contain  12  per  cent,  of  albumin,  prepare  a 
2   per  cent,   solution  by   suitable  dilution   with   distilled   water. 
Shake  thoroughly,  and  filter  through  a  plug  of  cotton. 

5.  Coagulation  by  Heating. — Heat  a  little  of  the  albumin  solu- 
tion in  a  test  tube.     Compare  the  coagulation  so  obtained  with 

*  A  mixture  of  casein  and  dry  egg  albumin. 


that  obtained  when  the  solution  is  diluted  (a)  20  times  with  dis- 
tilled water,  (b)  20  times  with  .1  per  cent,  salt  solution,  (c)  20 
times  with  .01  per  cent,  acetic  acid,  (d)  20  times  with  equal 
volumes  of  .1  per  cent,  salt  solution  and  .01  per  cent,  acetic  acid 
solution. 

6.  Coagulation  Temperature. — Ascertain  the  temperature  of 
coagulation  of  albumin  as  follows :     Faintly  acidify  a  portion  of 
the  solution  with  a  few  drops  of  .5  per  cent,  acetic  acid.    Filter 
if  necessary.    Place  a  portion  of  the  solution  in  a  test  tube,  insert 
a  thermometer  by  means  of  a  cork,  and  suspend  the  tube  in  a 
large  beaker  of  water.     Heat  the  beaker  slowly  with  a  small 
flame,  and  observe  the  point  at  which  the  albumin  solution  be- 
comes cloudy. 

7.  Sulphosalicylic  Acid  Test. — To  some  albumin  solution  in  a 
test  tube  add  a  few  drops  of  sulphosalicylic  acid  solution  (25  per 
cent.).     Determine  the  delicacy  of  the  test. 

8.  Nitric  Acid  Test. — Put  5  c.c.  of  the  solution  in  a  test  tube, 
and  introduce  5  c.c.  of  concentrated  nitric  acid  very  carefully 
with  a  pipet  to  the  bottom,  forming  an  under  layer.    Determine 
the  lowest  protein  concentration  at  which  the  test  is  unmistakable. 
Allow  10  minutes  if  the  reaction  is  slow  in  appearing. 

9.  Picric  Acid  Test. — Add  to  a  portion  of  the  albumin  solu- 
tion a  few  drops  of  a  solution  of  picric  acid  (i  per  cent.)  and 
citric  acid  (2  per  cent.) — Esbach's  reagent.    Determine  the  low- 
est protein  concentration  at  which  this  test  is  unmistakable. 

10.  Action  of  Ammonium  Sulphate. — Add  some  solid  ammo- 
nium sulphate  to  10  c.c.  of  the  albumin  solution  in  a  test  tube, 
shaking   frequently  until  the  solution  is  thoroughly   saturated. 
Allow  to  stand  for  a  while,  occasionally  shaking,  filter,  and  test 
the  filtrate  for  albumin  by  the  heat  test.     Test  the  solubility  in 
water  of  the  precipitate  on  the  filter  paper. 

11.  Action  of  Magnesium  Sulphate. — Perform  a  similar  ek- 
periment,  using  solid  magnesium  sulphate  instead  of  ammonium 
sulphate.    To  a  portion  of  the  filtrate  add  one  or  two  drops  acetic 
acid. 

12.  Biuret  Test. — To  a  portion  of  the  albumin  solution  add 

69 


a  little  sodium  hydrate,  then,  drop  by  drop,  very  dilute  copper 
sulphate.  The  solution  becomes  violet.  Study  the  delicacy  of 
the  reaction.  After  adding  to  the  albumin  solution  some  solid 
ammonium  sulphate,  repeat  the  test  with  (a)  the  same  amount 
of  alkali  (b)  a  large  amount  of  40  per  cent,  alkali. 

13.  Millon's  Test. — To  a  portion  of  the  albumin  solution  add  a 
few  drops  of  Millon's  reagent.     A  precipitate  forms,  which,  on 
heating,  becomes  brick  red.     Repeat,  using  a  dilute  solution  of 
phenol  instead  of  albumin.    On  what  group  in  the  protein  mole- 
cule does  this  test  depend?    Add  sodium  chlorid  and  repeat  the 
test.     Explain. 

14.  Xanthoproteic  Test. — To  a  few  c.c.  of  the  solution  add 
one-third  of  its  volume  of  concentrated  nitric  acid;  a  white  pre- 
cipitate may  or  may  not  be  produced  (according  to  the  concentra- 
tion and  the  nature  of  the  protein).     Boil.     The  precipitate  or 
liquid  turns  yellow.     Allow  the  solution  to  cool,  and  add  an 
excess  of  ammonia.    Explain. 

15.  The  Glyoxylic  Acid  Reaction  (Hopkins  and  Cole)  .—Treat 
2  or  3  c.c.  of  the  solution  with  the  same  volume  of  "reduced 
oxalic  acid."    Mix  and  add  an  equal  volume  of  concentrated  sul- 
phuric acid,  pouring  down  the  side  of  the  tube.     A  purple  ring 
forms  at  the  junction  of  the  fluids.     Mix  the  fluids  by  shaking 
the  tube  gently   from  side  to  side.     The  purple  color  spreads 
through  the  whole   fluid.     Repeat  in  the  presence  of  nitrates, 
chlorates,  nitrites,  excess  of  chlorids,  and  carbohydrates,  respec- 
tively. 

"Reduced  oxalic  acid"  is  prepared  by  Benedict's  method  as  follows : 

To  10  g.  powdered  magnesium  in  a  flask  add  a  little  water,  and 

then  add  slowly,  with  shaking  and  cooling,  250  c.c.  of  cold  saturated 

oxalic  acid  solution.    Filter,  acidify  the  filtrate  with  acetic  acid,  and 

dilute  to  one  liter. 

16.  Acetic  Acid  and  Potassic  Ferrocyanid.  — Acidify  some  albu- 
min solution  in  a  test  tube  with  acetic  acid,  and  add  a  few  drops 
of  potassic  ferrocyanid  solution.     A  white  flocculent  precipitate   is 
formed.     Determine  the  delicacy  of  this  precipitation. 

17.  Alcohol. — Add  an  excess  of  alcohol   (one  or  two  volumes) 
to  some  albumin  solution.    If  the  precipitate  is  small,  add  a  little  di- 
lute sodium  chlorid  solution. 

71 


H 


18.  Tannic  Acid. — Make  some  protein  solution  slightly  acid  with 
.1  per  cent,  of  acetic  acid,  and  add  a  few  drops  of  tannic  acid  solution. 

19.  Phosphotungstic  Acid. — Make  a  protein  solution  acid  with 
dilute  hydrochloric  acid,  and  add  a  few  drops  of  the  reagent. 

Globulins. — The  tests  are  made  upon  blood  serum. 

20.  Action  of  Carbon  Dioxid. — Dilute  5  c.c.  of  clear  serum  with 
45  c.c.  of  ice-cold  water.    Place  the  mixture  in  a  cylinder  or  large 
test  tube,  and  pass  through  it  a  stream  of  carbon  dioxid.    What  is 
the  effect  of  too  much  carbon  dioxid? 

21.  Precipitation  by  Dialysis. — Pour  20  c.c.  of  serum  into  a 
parchment  dialyzing  tube  previously  soaked  in  distilled  water. 
Suspend  the  tube,  with  its  contents,  in  a  large  volume  of  water. 
Explain  the  precipitation. 

Pour  serum  drop  by  drop  into  a  large  volume  of  distilled  water 
(in  a  beaker).  What  takes  place?  Explain. 

22.  Precipitation  by  Magnesium  Sulphate. — Saturate  about  5 
c.c.  of  the  serum  with  magnesium  sulphate.    A  heavy  precipitate 
will  be  formed.    Compare  this  with  the  action  of  the  same  salt 
on  the  egg-albumin  solution. 

23.  Precipitation  with  Ammonium  Sulphate. — To  30  c.c.  of 
serum  add  an  equal  volume  of  a  saturated  solution  of  ammonium 
sulphate,  thus  obtaining  a  half-saturated  solution.     Filter  off  the 
precipitate,  wash  two  or  three  times,  with  a  half-saturated  ammo- 
nium sulphate  solution,  and  dissolve  in  about  60  c.c.  of  water. 
This  yields  a  clear  solution  of  paraglobulin.     Apply  5  protein 
tests  to  this  solution. 

24.  Keratin. — Show  that  keratin  (hair  or  horn)  is  a  protein. 

25.  Gelatin. — Make  dilute  gelatin  solution,  and  with  it  make 
6  tests  for  protein  (including  Millon's).    Test  for  sulphur. 

26.  Phosphoproteins. — Test  the  solubility  of  casein  in  water, 
dilute  acid,  dilute  alkali,  and  dilute  salt  solution. 

Make  six  protein  tests  on  a  solution  of  casein.  How  would 
you  test  for  albumin  and  casein  when  both  are  present?  Apply 
to  milk  and  to  unknown  furnished. 

27..  Peptones  (Proteoses) . — For  the  following  experiments  use 
the  peptic  digestion  mixture  obtained  with  the  pig  stomach  (p. 

73 


— ).  Filter,  carefully  neutralize,  heat  to  boiling  (why?),  and 
again  filter.  Use  the  filtrate. 

To  a  small  portion  add  2  or  3  drops  of  dilute  acetic  acid  and 
a  few  c.c.  of  saturated  sodium  chlorid  solution.  Study  the  effect 
of  heating  and  cooling  on  this  precipitate. 

Apply  protein  tests  described  under  "albumin,"  and  record  the 
results  obtained. 

Dialyze  about  10  c.c.  of  the  peptone  solution  against  about  100 
c.c.  of  distilled  water  in  a  beaker.  After  24  hours  test  the  out- 
side water  for  peptones.  Explain. 

28.  Ammo-acids,  Tyrosin  and  Leucin. — For  this  experiment 
use  the  pancreatic  digestion  mixture  prepared  for  the  study  of 
ferment  reactions  (p.  27). 

With  a  pipet  take  out  10  c.c.  of  clear  supernatant  liquid.  Filter 
this  portion  if  necessary ;  dilute  it  with  2  volumes  of  water,  and 
by  means  of  Mett's  tubes  determine  whether  the  proteolytic  fer- 
ment has  been  destroyed  or  is  still  active. 

Pour  the  rest  of  the  digestion  mixture  without  filtering  into  a 
good-sized  beaker  or  flask.  With  continuous  stirring  or  gentle 
shaking  to  prevent  burning  and  bumping,  heat  the  digestion  mix- 
ture until  it  begins  to  boil.  Some  care  is  needed  in  this  opera- 
tion because  of  the  presence  of  alcohol.  When  the  mixture  is 
boiling  remove  the  flame.  Note  approximately  the  volume  of  the 
mixture,  and  measure  into  a  test  tube  some  "Merck's  dialyzed 
iron,"  8-10  c.c.  for  each  100  c.c.  of  digestion  mixture.  Dump 
the  colloidal  iron  into  the  digestion  mixture,  and  shake  or  stir 
vigorously.  Filter. 

To  the  filtrate  add  a  few  drops  of  ammonia  and  5-10  g.  of 
bone-black.  Boil  for  a  few  minutes  and  again  filter.  A  clear, 
faintly-colored  solution  should  be  obtained. 

Pour  this  last  filtrate  into  an  evaporating  dish,  acidify  with  a 
little  acetic  acid,  and  boil  down  to  about  one-sixth  of  the  original 
volume. 

Transfer  the  concentrated  liquid  to  a  flask  or  beaker,  and  set 
aside  in  a  cool  place  for  a  day  or  two.  Tyrosin  and  leucin  crys- 
tallize out,  the  former  first  and  in  much  greater  abundance. 
Examine  the  sediment  under  the  microscope. 

The  isolation  of  other  amino-acids  from  the  mother  liquor  is 
much  more  difficult. 

75 


29.  Preparation  of  Cystin  (from  Wool). — Heat  50  g.  of  wool 
in  a  500  c.c.  flask  with  100  c.c.  concentrated  hydrochloric  acid 
on  a  waterbath  until  dissolved.  A  3-foot  glass  tube  should  be 
inserted  to  prevent  the  loss  of  too  much  acid  liquid.  When  dis- 
solved boil  very  gently  over  a  small  flame  for  3-4  hours.  Add 
solid  sodic  acetate  (100-130  g.)  until  no  free  mineral  acid  can  be 
detected  in  the  solution  by  means  of  congo  red  paper.  Allow  the 
mixture  to  stand  for  3-5  days.  The  longer  the  mixture  is  allowed 
to  stand,  up  to  3  weeks,  the  more  cystin  is  obtained.  Filter  on  a 
Buchner  funnel  and  wash  with  cold  water.  Then  dissolve  the 
precipitate  in  water  (150  c.c.)  plus  5-10  c.c.  concentrated  hydro- 
chloric acid,  add  about  20  g.  purified  bone-black,  and  boil  5-10 
minutes. 

To  prepare  pure  bone-black,  let  the  impure  sample  stand  in  an 
excess  of  dilute  hydrochloric  acid  over  night,  filter,  and  wash  with 
cold  water  until  the  filtrate  is  neutral. 

Filter  again  with  suction,  heat  the  filtrate  to  boiling,  and  neu- 
tralize the  hot  hydrochloric  acid  by  adding  very  slowly  hot  con- 
centrated sodic  acetate  solution  (avoid  an  excess,  test  with  congo 
red  paper).  The  precipitate  formed  consists  of  cystin,  and  should 
be  very  white  and  pure.  If  it  is  dark  colored,  re-dissolve  in 
water  and  a  little  hydrochloric  acid,  and  repeat  the  bone-black 
treatment. 

Keep  the  mixture  boiling,  and  add  very  slowly  the  hot  sodic 
acetate  solution  until  the  crystallization  begins ;  keep  hot,  and  after 
a  few  minutes  add  cautiously  a  little  more  acetate.  Well-formed, 
large,  and  characteristic  crystals  should  be  obtained. 

Examine  the  crystals  under  the  microscope. 

Test  for  sulphur.  Test  also  for  ty rosin.  (Ordinarily  the 
crystals  are  quite  free  from  tyrosin.) 

Tyrosin  can  be  prepared  from  the  original  mother  liquor  by 
decolorizing  with  bone-black  and  letting  it  stand  in  a  cold  place. 


77 


PART  VI 
TJKINE  ANALYSIS  AND  METABOLISM 

Each  student  is  expected  to  keep  on  hand  accurately  collected  and 
measured  twenty-four  hour  specimens  of  his  own  urine.  The  urines 
should  be  preserved  by  the  addition  of  5  c.c.  of  chloroform  to  each 
bottle.  All  determinations  should  be  made  in  duplicates. 

1.    Aeration  Method  for  the  Determination  of  Ammonia. — 

Measure  25  c.c.  of  the  ammonium  sulphate  solution  previously 
used  for  nitrogen  determinations  (p.  17)  into  a  tall  aerometer 
cylinder.  The  cylinder  is  fitted  with  a  two-hole  rubber  stopper 
and  glass  connections  so  arranged  that  compressed  (outside)  air 
(or  laboratory  air  previously  freed  from  ammonia)  is  passed  to 
the  bottom  of  the  cylinder  and  out  through  a  calcium  chlorid 
tube  filled  with  dry  cotton,  and  then  through  a  special  absorption 
tube,  into  a  receiver  containing  water  and  a  known  amount  of 
acid.  The  cotton  serves  the  purpose  of  holding  back  traces  of 
solid  sodic  carbonate,  formed  by  evaporation  on  the  sides  of  the 
cylinder. 

Add  to  the  ammonium  sulphate  solution  about  10  g.  sodium 
chlorid,  about  2  g.  sodic  carbonate,  and  a  few  drops  of  kerosene. 
Do  not  add  any  water ;  the  greater  the  volume  the  longer  it  takes 
to  drive  off  all  the  ammonia.  Pass  a  very  strong  air  current 
through  the  mixture  for  one  and  one-half  hours,  and  collect  the 
ammonia,  which  the  air  carries  off,  in  a  receiver  containing  25  c.c. 
.1  N  acid  and  about  200  c.c.  water.  Titrate,  and  compare  the 
result  with  the  figures  obtained  by  distillation  (p.  21).  If  the 
results  are  too  low,  the  air  current  has  been  too  slow,  or  the  aera- 
tion process  has  not  been  continued  long  enough  to  drive  off  all 
the  ammonia. 

In  a  similar  manner  determine  the  ammonia  in  urine  (25  c.c.) 
and  calculate  the  24-hour  amount.  When  working  with  urine  it  is 
desirable,  though  not  absolutely  necessary,  to  substitute  10  g.  of 

79 


potassium  oxalate  for  the  10  g.  of  sodium  chlorid.  Salts  hasten 
the  removal  of  the  ammonia,  and  oxalate  incidentally  prevents 
the  (possible)  formation  of  insoluble  ammonium  magnesium 
phosphate. 

Save  the  remainder  of  the  ammonium  sulphate  solution  for 
sulphate  determinations. 

2.  Colcgimetric  Method  for  the  Determination  of  Ammonia. — 
With  an  Ostwald  pipet  measure  I  or  2  c.c.  of  the  amnioi\ium 
sulphate  solution  into  a  large  Jena  test  tube  (200  x  25  mm.). 
Choose  the  amount  which  contains  nearer  I  mg.  of  nitrogen.  Fit 
the  test  tube  with  a  two-hole  rubber  stopper  carrying  an  inlet 
tube,  reaching  to  the  bottom,  and  an  outlet  tube.  Connect  the 
former  with  the  compressed  air  jet,  and  the  latter  with  an  ab- 
sorption tube  having  small  holes  drilled  through  the  wall  at  the 
end.  Insert  the  absorption  tube  into  a  100  c.c.  measuring  flask 
containing  20-30  c.c.  of  distilled  water  and  2  c.c.  .1  N  HC1.  Add 
2  drops  kerosene  and  a  few  drops  of  a  solution  containing  po- 
tassium oxalate  and  potassium  carbonate  (15  per  cent,  of  each), 
quickly  put  the  stopper  firmly  in  the  tube,  and  start  the  air  cur- 
rent, gradually  increasing  its  speed  for  about  two  minutes.  In 
ten  minutes  all  the  ammonia  should  have  been  driven  over  into 
the  receiving  flask. 

Remove  the  absorption  tube,  rinsing  it  with  water,  and  dilute 
the  contents  in  the  flask  to  about  60  c.c. 

Pipet  I  c.c.  of  standard  ammonium  sulphate,  using  the  Ostwald 
pipet,  into  another  100  c.c.  measuring  flask,  and  dilute  with  water 
to  60  c.c. 

Nesslerize  both  solutions  simultaneously  in  the  following  man- 
ner: Provide  two  small  beakers  (100  c.c.), -and  place  20-25  c.c. 
of  distilled  water  in  each.  Add  to  each  5  c.c.  of  Nessler's  reagent^ 
Mix  the  reagent  with  the  water,  and  add  it  immediately  to  the 
ammonia  solutions  (about  one-third  of  the  diluted  Nessler  reagent 
at  a  time).  Shak«  after  each  addition. 

Fill  both  flasks  up  to  the  mark  with  distilled  water  and  mix. 
The  resulting  deep  red  colloidal  solutions  must  be  absolutely 
crystal  clear.  If  the  least  turbidity  or  opalescence  is  perceptible 
in  either  flask,  that  flask  must  be  discarded.  When  properly 
made  the  Nesslerized  solutions  remain  clear  and  unchanging  for 
several  hours. 

The  colorimetric  value  of  the  unknown  solution  is  next  deter- 

81 


mined  by  means  of  a  Duboscq  colorimeter.  In  this  process  the 
standard  solution,  set  at  20  mm.  in  the  colorimeter  cups,  must 
first  be  read  against  itself.  The  readings  obtained  in  such  com 
parisons  should  come  within  .2  mm.  of  the  theoretical  figure 
(20  mm.).  When  the  instrument  (and  the  eye)  are  thus  adjusted, 
rinse  out  the  contents  of  one  of  the  colorimeter  cups  with  the 
unknown  solution,  and  repeat  the  color  comparison,  taking  from 
3  to  6  separate  readings.  The  ammonia  content  is  inversely  pro- 
portional to  the  depth  of  the  color.  Twenty  divided  by  the  read- 
ing gives  the  ammonia  nitrogen  (in  mg.)  in  the  volume  of  ammo- 
nium sulphate  solution  taken.  Calculate  the  result  and  compare 
with  the  figures  previously  obtained  (a)  by  distillation  (p.  21), 
(b)  by  the  macro-aeration  process  (p.  79). 

Repeat  the  ammonia  determination  described  above,  with  urine. 
The  volume  of  urine  taken  should  give  .75-1.25  mg.  of  ammonia 
nitrogen  (1-5  c.c.).  Calculate  the  24-hour  quantity  of  the  am- 
monia thus  obtained,  and  compare  with  the  figure  obtained  by 
the  preceding  method. 

3.  Clinical  Method  for  the  Determination  of  Ammonia. — The 
special  reagents  required  in  this  determination  are  (a)  saturated 
potassium   oxalate  solution   and    (b)    formalin;   both   of   which 
must  be  neutral  to  phenolphthalein.    To  each  reagent  add  a  little 
of  the  indicator  and  .1  N  alkali  to  a  faint  pink  coloration. 

To  25  c.c.  of  urine  add  about  5  c.c.  of  the  neutralized  oxalate 
solution  and  2-3  drops  phenolphthalein  solution.  Titrate  the 
acidity  of  the  urine  to  a  faint  but  unmistakable  end  point.  Then 
add  about  5  c.c.  of  the  neutralized  formalin  and  again  titrate  to 
the  same  degree  of  coloration  as  in  the  preceding  titration.  Each 
c.c.  of  the  .1  N  alkali  used  in  this  titration  corresponds  to  I  c.c. 
.1  N  ammonia. 

The  formaldehyd  combines  with  the  ammonia  giving  neutral 
hexamethylene  tetramin,  thus  setting  free  acid  equivalent  to  the 
amount  of  ammonia  present. 

4.  Total  Nitrogen.   Colorimetric  Method. — Special   equipment 
called  for:      (a)    Duboscq  colorimeter,    (b)    standardized   I   c.c. 
"Ostwald  pipets,"  with  extra  long  stems,  (c)  a  solution  of  spe- 
cially purified  ammonium  sulphate,  of  such  strength  that  I  c.c. 
contains  I  mg.  of  nitrogen  (4.7062  g.  salt  per  liter),  (d)  modified 
Nessler-Winkler  reagent  (see  p.  161). 

83 


Consult  the  instructor  before  beginning  this  determination. 

Dilute  the  urea  solution  previously  used  for  N  determinations 
(p.  21 )  so  that  i  c.c.  contains  approximately  I  mg.  of  nitrogen. 

Pipet  i  c.c.  of  the  diluted  urea  solution,  using  the  Ostwald  pipet, 
into  a  large  Jena  test  tube.  Add  i  c.e.  concentrated  H2SO4,  i  g. 
K2SO4,  i  drop  CuSO4  solution  (10  per  cent.),  and  a  small  pebble 
or  glass  bead.  •  - 

Boil  over  a  micro-burner  for  10  minutes.  Allow  to  cool  until 
the  digestion  mixture  just  begins  to  become  viscous  (it  must  not 
solidify)  ;  then  add  about  6  c.c.  of  water,  at  first  a  few  drops 
at  a  time,  then  more  rapidly  if  the  digestion  mixture  begins  to 
solidify. 

Draw  up  3  c.c.  of  saturated  sodic  hydrate  solution  into  the  long 
glass  tube  carried  in  the  two-hole  rubber  stopper,  and  retain  by 
means  of  a  piece  of  rubber  tubing  and  a  pinchcock.  Stopper  the 
test  tube  with  this  apparatus,  and  connect  the  outlet  tube  to  an 
absorption  tube  having  several  small  holes  drilled  through  the 
wall  at  the  end.  The  absorption  tube  is  placed  in  a  100  c.c. 
measuring  flask,  containing  20-30  c.c.  of  distilled  water  and  2-5 
c.c.  tenth  normal  HC1. 

Release  the  pinchcock,  thus  allowing  the  alkali  to  flow  into 
the  test  tube.  Connect  with  "the  compressed  air,  and  turn  on 
carefully  so  that  one  or  two  bubbles  mix  the  solutions.  Run  the 
air  slowly  for  two  minutes,  then  rapidly  for  ten  minutes,  just 
as  in  the  ammonia  determination.  At  the  end  of  this  time  all 
of  the  ammonia  should  have  gone  over. 

Remove  the  delivery  tube,,  rinsing  it  with  water,  and  dilute 
the  contents  in  the  flask  to  about  60  c.c.  Complete  the  determina- 
tion as  in  the  case  of  ammonia. 

Calculate  the  results,  and  compare  with  the  figures  obtained  by 
Kjeldahl's  process  (p.  22). 

Repeat  the  determination  with  diluted  urine  (1-5  or  i-io). 
Compare  with  a  Kjeldahl  determination  (5  c.c.  urine)  upon  this 
same  urine  undiluted. 

Save  the  original  urea  solution  for  direct  urea  determinations. 

5.  Reactions  of  Urea. — Put  a  crystal  of  urea  on  a  glass  slide 
or  a  watch-glass  and  cover  it  with  a  drop  of  water.  With  a  glass 
rod,  put  a  drop  of  nitric  acid  next  to  this.  Let  the  two  drops 
run  together,  and  notice  the  precipitation  of  urea  nitrate  at  the 

85 


junction.    Examine  under  the  microscope,  and  sketch  the  crystals. 

Put  a  few  crystals  of  urea  into  a  dry  test  tube,  and  heat  till 
they  melt.  With  moist  litmus  paper  test  the  reaction  of  the  fumes 
given  off.  Explain. 

Cool  the  test  tube.  To  the  residue,  consisting  of  biuret  and 
cyanuric  acid,  add  a  little  water,  filter,  and  with  the  filtrate  make 
the  biuret  test. 

Dissolve  a  few  crystals  of  urea  in  5  c.c.  water  in  a  test  tube. 
Test  its  reaction  with  litmus  paper.  Heat  the  solution  to  boiling 
and  test  the  steam  with  moist  litmus  paper.  Cool  the  liquid  and 
test  with  litmus  paper.  Explain. 

To  a  solution  of  urea  in  a  test  tube  add  an  equal  volume  of 
sodium  hypobromite  solution.  Make  this  by  mixing  and  cooling 
equal  volumes  of  bromin  solution  and  40  per  cent,  sodic  hydrate 
solution. 

The  reaction  with  sodium  hypobromite  has  been  used  for  the 
quantitative  determination  of  urea,  but  as  ordinarily  used  for  this 
purpose  the  method  has  very  little  value. 

6.  Colorimetric  Method  for  the  Determination  of  Urea  (7.  Biol. 
Chem.,  II,  507). — Measure  i  c.c.  of  the  urea  solution  used  for 
nitrogen  determinations  (diluted  1-5  or  i-io)  into  a  large  dry 
Jena  test  tube.  Add  I  c.c.  50  per  cent,  acetic  acid,  7  g.  dried 
potassium  acetate,  a  pebble,  and  a  temperature  indicator  (de- 
scribed below).  Insert  through  a  rubber  stopper,  as  condenser, 
a  special  long  calcium  chlorid  tube.  Then  heat  with  a  micro- 
burner  until  a  clear  solution  is  obtained  and  the  indicator  melts. 
This  should  happen  in  two  or  three  minutes.  Add  water  first 
through  the  condenser,  and  then  directly  into  the  tube  after  tak- 
ing out  the  rubber  stopper.  The  sides  of  the  tube  should  be 
rinsed  carefully  without  using  more  than  5  or  6  c.c.  of  water, 
without  letting  the  contents  in  the  test  tube  solidify,  and  with- 
out losing  ammonia  (which  will  occur  if  the  stopper  is  taken 
out  too  soon). 

Add  to  the  contents  of  the  tube  about  2  c.c.  30-40  per  cent, 
caustic  soda  and  drive  out  the  ammonia,  as  in  the  total  nitrogen 
determination  with  the  air  current.  Jf  the  total  volume  in  the 
test  tube  does  not  exceed  10  c.c.  the  ammonia  will  go  over  in  10 
minutes.  Nesslerize  and  compare  with  standard  ammonium  sul- 
phate as  before. 

87 


The  temperature  indicator  is  HglCl,  a  mixed  halid  prepared  by 
heating  together  equivalent  quantities  of  mercuric  chlorid  and  mer- 
curic iodid  to  150-160°  C,  for  3-4  hours.  This  compound  melts  at 
153°  C.  It  cannot  be  purified,  hence  the  salts  taken  should  be 
weighed  out  with  care.  .I-.2  g.  of  the  compound  is  sealed  up  in 
small  glass  bulbs. 

7.  Marshall's  Urease  Method  for  the  Determination  of  Urea.— 

PREPARATION  OF  ENZYME. — Grind  25  g.  of  soy  beans  in  a  coffee 
mill.  Extract  the  meal  with  125  c.c.  water  for  one  hour,  shaking 
frequently.  Add  to  the  mixture  about  25  c.c.  approximately  deci- 
normal  hydrochloric  acid  and  let  stand  for  5  minutes.  Filter 
with  suction  on  a  Buchner  funnel.  To  the  filtrate  add  5  c.c.  of  a 
solution  made  by  dissolving  70  g.  disodic  phosphate  and  27  g. 
monopotassium  phosphate  in  100  c.c.  of  water.  Shake  until  the 
phosphates  are  dissolved  and  dilute  to  a  volume  of  200  c.c.  In 
the  cold  this  enzyme  solution  keeps  its  strength  for  several  weeks. 

THE  DETERMINATION. — To  i  c.c.  of  urea  solution  or  urine 
(so  diluted  as  to  contain  about  I  mg.  urea-nitrogen  per  c.c.)  in  a 
large  test  tube  add  1-2  c.c.  of  the  enzyme  extract  (a  large  excess 
is  undesirable).  Add  2  drops  of  kerosene,  and  immediately  stop- 
per with  the  aeration  apparatus  used  for  ammonia  or  other  nitro- 
gen determinations.  Let  stand  for  15  minutes.  Now  pass  the 
current  through  the  mixture  for  about  half  a  minute  to  drive 
off  any  free  ammonia  which  may  be  present,  collecting  it  as 
usual  in  a  100  c.c.  volumetric  flask  containing  25  c.c.  water  and 
2  c.c.  decinormal  hydrochloric  acid.  Stop  the  air  current,  open 
the  tube,  and  add  about  I  c.c.  of  potassium  carbonate  oxalate 
solution  (as  in  the  preceding  method). 

Close  the  tube  promptly,  start  the  air  current  again,  gradually 
increasing  the  speed  and  running  it  very  fast  for  10-15  minutes. 

The  subsequent  Nesslerization  and  color  comparison,  with  I  mg. 
of  ammonia-nitrogen  as  the  standard,  are  made  in  the  usual 
manner. 

Repeat  the  urea  determination  on  two  twenty-four  hour 
urines.  Calculate  the  urea  nitrogen  and  urea  in  each  sample, 
making  due  allowance  for  the  ammonia  of  the  urine. 

8.  Uric  Acid  Preparation  of  Uric  Acid  from  Urine. — To  500 
c.c.  of  urine  add  25  g.  of  ammonium  sulphate,  stir  until  the  sul- 
phate is  dissolved,  and  add  about  10  c.c.  of  ammonia.    Let  stand 
over  night,  filter,  and  wash  two  or  three  times  with  water  con- 

89 


taining  a  little  ammonia.  Transfer  the  precipitate  to  a  small 
beaker,  add  a  few  drops  of  hydrochloric  acid,  and  let  stand  till 
the  following  day.  Examine  the  crystals  under  the  microscope. 

9.  Murexid  Test  for  Uric  Acid. — Place  a  few  uric  acid  crystals 
on  a  porcelain  crucible  cover.     Add  three  drops  of  strong  nitric 
acid.     Heat  cautiously,  blowing  on  the  liquid  to  complete  dry- 
ness.    A  red  color  should  appear.    Let  cool  and  add  a  few  drops 
of  dilute  ammonia.     Repeat  with  caffein.     Explain. 

10.  Phosphotungstic  Acid  Test  for  Uric  Acid. — Dissolve  a  few 
crystals  of  uric  acid  in  2  c.c.  very  dilute  sodic  hydrate  solution. 
Add  i  c.c.  of  "qualitative  uric  acid  reagent"  (see  p.  171).    Then 
add   10   c.c.   of   saturated   sodium  carbonate   solution.     A   pro- 
nounced blue  coloration  should  be  obtained.    Repeat  the  reaction 
with  5  c.c.  of  urine. 

11.  Volumetric  Determination  of  Uric  Acid. — Transfer  to  a 
small  weighed  beaker  exactly  500  mg.  of  pure  uric  acid  (Kahl- 
baum's).     Add  50  c.c.  of  a  .4  per  cent,  lithium  carbonate  solu- 
tion and  25-35  c-c-  °f  distilled  water.    Stir  until  dissolved.    When 
solution  is  complete,  transfer  the  solution  without  loss  of  a  single 
drop  to  a  500  c.c.  volumetric  flask.    Fill  to  the  mark  with  water 
and  mix.    This  alkaline  uric  acid  solution  will  usually  keep  with- 
out appreciable  decomposition  for  5-7  days. 

From  25  c.c.  of  the  alkaline  uric  acid  solution  prepare  a  dilute, 
acidified  (and  therefore  more  permanent)  standard  solution  of  uric 
acid  according  to  the  directions  given  on  p.  169.  Transfer  this  solu- 
tion, 20  c.c.  of  which  contains  I  mg.  of  uric  acid,  to  a  bottle,  label, 
and  preserve  for  use  in  connection  with  colorimetric  determinations 
of  uric  acid. 

.  Prepare  .05  N  potassium  permanganate  solution  by  dissolving 
1.578  g.  in  water  and  diluting  to  one  liter.  (Glass  stoppered  flask 
necessary.)  Standardize  this  solution  (in  terms  of  uric  acid) 
as  follows :  Measure  50  c.c.  of  the  lithium  carbonate  solution  of 
uric  acid  into  a  beaker,  add  20  c.c.  concentrated  sulphuric  acid, 
stir,  and  titrate  immediately  with  the  .05  N  potassium  perman- 
ganate solution.  The  very  first  coloration  extending  through- 
out the  liquid  marks  the  end  point  of  the  titration.  Calculate  the 


number  of  mg.  of  uric  acid  to  which  each  cubic  centimeter  of 
permanganate  is  equivalent. 

Measure  out  two  50  c.c.  samples  of  the  alkaline  uric  acid  solu- 
tion. Add  equal  volumes  of  water  to  each  and  then  stir  in  10  g. 
ammonium  sulphate  until  dissolved.  Add  about  5  c.c.  concen- 
trated ammonium  hydrate,  cover,  and  let  stand  for  twenty-four 
hours.  Filter.  Wash  the  precipitate  back  into  the  beaker  with 
about  100  c.c.  water.  Add  sulphuric  acid  and  titrate,  as  when 
standardizing  the  potassium  permanganate  solution. 

THE  DETERMINATION  OF  URIC  ACID  IN  URINE:  To  150  c.c. 
fresh  urine  add  37.5  c.c.  special  ammonium  sulphate  solution.* 
Let  stand  for  half  an  hour,  decant,  and  unless  perfectly  clear  fil- 
ter through  a  dry  filter.  Measure  out  125  c.c.  of  the  filtrate  (cor- 
responding to  100  c.c.  urine),  add  about  5  c.c.  ammonia,  and  let 
stand  for  48  hours.  Filter  and  wash  the  precipitate  five  or  six 
times  with  10  per  cent,  ammonium  sulphate  solution.  Wash  the 
precipitate  into  a  beaker  by  means  of  about  100  c.c.  water.  Add 
sulphuric  acid  and  titrate.  Calculate  the  twenty-four  hour  quan- 
tity of  uric  acid  and  of  uric  acid  nitrogen. 

12.  New  Colorimetric  Method  for  the  Determination  of  Uric 
Acid. — Because  of  the  fading  of  the  blue  color  produced  by  the 
action  of  the  phosphotungstic  acid  reagent  on  uric  acid,  the  colon- 
metric  quantitative  determination  has  now  been  revised  so  as  to 
permit  the  use  of  the  reagent  previously  used  only  for  phenol  deter- 
minations— the  "phenol  and  uric  acid  reagent.  The  revisions  involved 
are  quite  extensive. 

The  new  method  is  now  as  follows : 

Transfer  2-5  c.c.  of  urine,  according  to  concentration,  to  a  12 
c.c.  centrifuge  tube,  and  add  distilled  water  until  the  tube  con- 
tains about  6  c.c.  Add  5  c.c.  of  a  silver  lactate  solution  (silver 
lactate  5  per  cent.,  lactic  acid  5  per  cent.),  and  stir  the  mixture 
thoroughly  with  a  glass  rod.  Rinse  off  the  rod  with  a  few  drops 
of  distilled  water.  Centrifuge  the  counterbalanced  tube  for  2-3 
minutes.  Add  a  drop  of  the  silver  solution  to  the  clear  super- 
natant liquid;  if  a  precipitate  forms,  add  2  c.c.  more  of  the  silver 
solution  and  centrifuge  again ;  if  no  precipitate  forms,  pour  off 
the  liquid  as  completely  as  possible. 

*  The  solution  contains  50  per  cent,  ammonium  sulphate,  5  per  cent, 
uranium  acetate,  and  5  per  cent,  acetic  acid. 

93 


To  the  precipitate  in  the  centrifuge  tube  add,  from  a  buret, 
4  c.c.  of  a  5  per  cent,  sodium  cyanid  solution  (poisonous — j  c.c. 
may  be  fatal  dose}  and  about  5  c.c.  of  water.  Stir  the  mixture 
with  a  glass  rod  until  a  perfectly  clear  solution  is  formed.  Rinse 
the  rod,  collecting  the  rinsings  in  a  100  c.c.  graduated  flask;  pour 
the  contents  of. the  tube  into  the  same  flask;  and  rinse  the  tube 
twice  with  about  5  c.c.  of  water,  which  is  also  poured  into  the 
flask.  Add  water,  to  a  volume  of  about  40  c.c.,  and  4  c.c.  of  uric 
acid-phenol  reagent  (see  p.  171). 

In  another  100  c.c.  flask  place  20  c.c.  of  a  standard  uric  acid 
solution  (see  p.  169)  containing  i  mg.  of  uric  acid;  add  4  c.c. 
of  cyanid  solution,  about  20  c.c.  of  water,  and  4  c.c.  of  uric  acid- 
phenol  reagent.  Then  add  20  c.c.  of  20  per  cent,  sodium  car- 
bonate solution  to  each  flask.  At  the  end  of  20  minutes  fill  both 
flasks  up  to  volume,  and  read  against  each  other  in  a  colorimeter. 
If  a  precipitate  has  formed,  the  solutions  must  be  filtered  or 
centrifuged  before  making  the  color  comparison. 

Set  the  standard  uric  acid  solution  at  20  m.m.,  and  read  it 
against  itself  several  times  before  reading  the  unknown.  Twenty 
divided  by  the  reading  of  the  unknown  (in  m.m.)  gives  the 
amount  of  uric  acid  (in  mg.)  present  in  the  volume  of  urine  taken 
for  analysis. 

When  working  with  urines  containing  albumin,  the  albumin 
must  be  removed  by  coagulation  with  acetic  acid  and  filtering 
before  making  the  determination. 

13.  Creatinin. — Transfer  to  a  small  bottle  about  3  g.  dry  picric 
acid.  Add  about  100  c.c.  of  urine,  insert  a  cork,  and  shake  con- 
tinuously for  ten  minutes.  Filter  a  portion  of  the  mixture  and 
transfer  5  c.c.  of  the  filtrate  to  a  bottle  or  flask  (capacity  not  less 
than  250  c.c.). 

Measure  5  c.c.  of  the  original  urine  into  another  similar  flask 
or  bottle.  Add  to  each,  first  20  c.c.  saturated  picric  acid  solu- 
tion, and  then  5  c.c.  10  per  cent,  sodic  hydrate.  Let  stand  5-10 
minutes  and  add  200  c.c.  tap  water  to  each. 

Remove  by  decantation  the  liquid  from  the  bottle  containing 
urine  and  picric  acid,  and  to  the  sediment  add  about  25  c.c.  water 
and  15  c.c.  sodic  hydrate  (10  per  cent.).  Shake,  let  stand  for  a 
few  minutes,  and  fill  the  bottle  with  tap  water. 

The  substance  in  urine  which  is  precipitated  by  picric  acid  and 
which  gives  a  deep  red  color  with  alkaline  picrate  solutions, 

95 


is  creatinin.  No  other  known  substance  occurring  in  normal 
urine  gives  this  color  reaction.  Therefore,  on  the  basis  of 
this  reaction,  it  is  easy  to  determine  the  creatinin  quantita- 
tively. 

14.  Quantitative  Determination  of  Creatinin  (/.  Biol.  Chem., 
17,  469). — A  suitable  and  convenient  "creatinin  reagent"  is  pre- 
pared by  adding  75  c.c.  sodic  hydrate  (10  per  cent.)  to  a  liter 
of  saturated  picric  acid  solution.     The  solution  keeps  well  in 
tightly  stoppered  bottles. 

By  means  of  an  Ostwald  pipet  transfer  I  c.c.  of  urine  to  a 
100  c.c.  volumetric  flask.  To  another  similar  flask  transfer  I  c.c. 
of  a  standard  creatinin  solution  (1.61  g.  creatinin  zinc  chlorid  dis- 
solved in  a  liter  .1  N  hydrochloric  acid),  I  c.c.  of  which  contains 
i  mg.  creatinin.  To  each  flask  add  20  c.c.  of  the  alkaline  picrate 
solution  and  let  stand  for  ten  minutes.  Dilute  to  the  mark  with 
water  and  mix. 

Read  the  standard  against  itself  in  the  colorimeter  at  20  m.m. 
until  the  correct  value  (20  m.m.)  can  be  obtained  several  times  in 
succession.  The  error  in  reading  should  not  exceed  .2  m.m. 
Rinse  the  right-hand  cup  and  prism  with  the  unknown,  and 
determine  its  color  in  terms  of  the  standard  set  at  20  m.m. 
Twenty  divided  by  the  reading  gives  the  creatinin  in  milligrams 
.in  the  quantity  of  urine  taken  (1-5  c.c.). 

Calculate  the  total  twenty-four  hour  creatinin. 

15.  Creatin. — Unless  considerable  meat  or  fish  has  been  eaten 
the   urine   of    normal   adults    contains    only   traces    of    creatin. 
Urines  of  children  and  of  sick  persons,  particularly   fever  pa- 
tients, appear,  on  the  other  hand,  to  contain  relatively  consider- 
able quantities  of  creatin  (.2  g.  to  I  g.  or  more  per  day, in  fever 
patients). 

Creatin  is  determined  in  such  urines  by  first  converting  it  into 
creatinin.  This  is  done  as  follows : 

Measure  the  urine  (usually  i  c.c.)  into  a  flask  (capacity  300 
c.c.)  and  add  20  c.c.  saturated  picric  acid  solution  (not  the 
creatinin  reagent).  Weigh  flask  and  contents  and  add  about  150 
c.c.  water.  Boil  gently  for  45  minutes,  then  more  rapidly  until 
the  original  volume  (determined  by  weighing)  is  obtained  (a  vari- 
ation of  3  or  4  g.  makes  no  appreciable  difference).  Cool.  Add 
1.5  c.c.  10  per  cent,  sodic  hydrate,  let  stand  10  minutes,  and  com- 

97 


pare,  as  in  the  case  of  preformed  creatinin,  with  the  color  obtained 
from  i  mg.  creatinin. 

Twenty  divided  by  the  reading  gives  the  sum  of  the  creatin  and 
creatinin  present. 

Calculate  the  twenty-four  hour  quantity  and  subtract  the  pre- 
formed creatinin. 

If  an  autoclave  is  available,  the  conversion  into  creatin  can 
be  made  more  rapidly. 

Measure  the  urine  (i  c.c.)  into  a  100  c.c.  volumetric  flask, 
and  add  20  c.c.  saturated  picric  acid  solution.  Cover  the  mouth 
of  the  flask  with  tinfoil,  and  heat  in  the  autoclave  at  H5°-i2O° 
for  20  minutes.  Cool,  add  1.5  c.c.  sodic  hydrate,  and  finish  the 
determination  in  the  usual  manner. 

16.  Hippuric  Acid. — Take  with  the  evening  meal  2  g.  of  so- 
dium benzoate,  and  collect  the  urine  until  next  morning. 

Evaporate  to  small  volume  and  transfer  with  a  little  wash 
water  to  a  small  flask.  Acidify  strongly  with  sulphuric  acid  and 
put  away  for  twenty-four  hours.  Filter  and  dry  the  precipitate, 
consisting  of  hippuric  acid,  uric  acid,  and  other  substances.  Ex- 
tract the  hippuric  acid  with  acetic  ether.  Set  aside  for  sponta- 
neous evaporation.  Examine  microscopically.  Heat  the  dry  sub- 
stance in  a  dry  tube,  and  note  the  odor  of  bitter  almonds  (benzal- 
dehyd). 

17.  Determination  of  Inorganic  Sulphates   (/.  Biol.  Ckem., 
i,   131). — Measure  into   a  small  flask  or  beaker  25  c.c.  of  the 
ammonium  sulphate  solution  in  which  the  ammonia  was  deter- 
mined  (p.  21 ).     Dilute  with  water  to  about  100  c.c.     Add  10 
c.c.  20  per  cent,  sodium  chlorid  solution   (free  from  sulphate), 
5  c.c.  concentrated  hydrochloric  acid,  and  finally,  drop  by  drop 
(without   shaking),   about    15   c.c.    5   per   cent,   barium   chlorid 
solution.     Let  stand  for  one  hour,  and  filter  through  a  previously 
prepared  Gooch  crucible  (consult  the  instructor).    Wash  about  a 
dozen  times  with  cold  water,  ignite,  and  weigh.    From  the  weight 
of  BaSO4  obtained,  calculate  the  ammonium  sulphate,  and  com- 
pare with  the  weight  of  ammonium  sulphate  used  in  making  up 
the  original  solution. 

18.  Determinations  of  Total  Sulphates, — Repeat  the  determina- 
tion, omitting  the  addition  of  sodium  chlorid,  and  heating  the 

QQ 


solution  to  boiling   for   15  minutes  before   adding  the  barium 
chlorid. 

Repeat  the  two  sulphate  determinations  described,  with  25  c.c. 
of  urine,  but  omitting  the  use  of  sodium  chlorid.  Explain  the 
results. 

19.  Determination  of  Total  Sulphur  (Benedict,  J.  Biol.  Chem., 
6,  363;  W.  Dems,  J.  Biol.  Ghent.,  8,  401). — To  25  c.c.  of  urine 
contained  in  a  porcelain  evaporating  dish  (10-12  cm.  diameter) 
add  exactly  5  c.c.  of  a  solution  containing  25  per  cent,  copper 
nitrate,  25  per  cent,  sodium  chlorid,  and  10  per  cent,  ammonium 
nitrate.     Evaporate  to  dryness  on  the  water-bath.     Then  heat 
over  a  flame,  gradually  increasing  the  heat  until  the  dish  is  red 
hot,  and  continue  heating  for  10  to  15  minutes.     Allow  to  cool. 
Add  20  c.c.  dilute  hydrochloric  acid  and  warm  gently.     Rinse 
into  a  flask  or  beaker  by  means  of  about  100  c.c.  hot  water.    Heat 
to  boiling,  and  add  drop  by  drop  25  c.c.  of  a  10  per  cent,  barium 
chlorid  solution.     Filter,  wash,  ignite,  and  weigh. 

20.  Phosphates. — Add  a  few  drops  neutral  calcium  chlorid  so- 
lution : 

(a)  to  5  c.c.  i  per  cent,  monopotassium  phosphate  solution, 

(b)  to  5  c.c.  I  per  cent,  disodium  phosphate  solution, 

(c)  to  a  mixture  of  the  two  phosphate  solutions, 

(d)  to  5  c.c.  turbid  urine, 

(e)  to  5  c.c.  clear  urine, 

(f)  to  5  c.c.  of  urine  after  filtering  off  the  precipitate  ob- 
tained by  the  addition  of  a  little  ammonia.    Explain  the  results. 

21.  Determination  of  Phosphates. — STANDARD  PHOSPHATE  SO- 
LUTION.— Dissolve   4.39   g.   pure   monopotassium   phosphate   in 
water  and  dilute  to  500  c.c.    Each  c.c.  contains  2  mg.  phosphorus. 
Label  and  preserve. 

STANDARD  URANIUM  SOLUTION. — Dissolve  18  g.  uranium  ace- 
tate and  50  c.c.  50  per  cent,  acetic  acid  in  water.  Dilute  to  500 
c.c.  If  turbid,  allow  to  settle  (for  a  day  or  two),  and  remove 
the  clear  supernatant  solution  by  means  of  a  siphon. 

Transfer  25  c.c.  of  the  phosphate  solution  to  a  flask,  add  5  c.c. 
special  sodic  acetate  solution  (containing  10  per  cent,  acetate  and 
3  per  cent,  acetic  acid),  heat  to  boiling,  and  add  15-20  c.c.  of  the 
clear  uranium  solution  from  a  buret.  Heat  again  to  boiling,  and 

101 


now  add  the  uranium  slowly  until  2  drops  of  the  phosphate 
uranium  mixture  when  added  (by  means  of  a  glass  tube  drawn 
out  to  a  point  like  a  pipet)  to  a  minute  pinch  of  powdered  potas- 
sium ferrocyanid  (on  a  white  plate)  gives  a  faint  yet  unmis- 
takable brownish  coloration. 

Repeat  until  the  exact  titrating  value  of  the  uranium  solution 
has  been  ascertained. 

Calculate  the  value  of  the  uranium  solution  in  terms  of  phos- 
phorus (P).  Label  and  preserve. 

DETERMINATION  OF  PHOSPHATES  IN  URINE. — Measure  50  c.c. 
of  urine  into  a  flask,  add  sodic  acetate,  heat  to  boiling,  and  titrate 
with  the  uranium  solution,  exactly  as  in  the  case  of  the  standard 
phosphate  solution.  After  having  found  the  approximate  phos- 
phate content  by  means  of  the  preliminary  titration,  repeat,  adding 
nearly  all  the  required  uranium  solution  at  once  to  the  hot  urine, 
and  finish  by  adding  only  a  few  drops  at  a  time. 

Calculate  in  terms  of  phosphorus  and  of  phosphoric  acid  the 
phosphate  content  of  the  24  hour  urine  under  examination. 

22.  Acidity  of  Urine. — Titrate,  with  phenolphthalein  as  indi- 
cator, the  acidity  of  the  monopotassium  phosphate  solution  used 
for  standardizing  the  uranium  solution. 

Repeat  after  having  added  5  c.c.  of  neutral  calcium  chlorid  so- 
lution (2  per  cent.). 

Repeat  after  having  added  5  c.c.  of  the  calcium  chlorid  solution 
and  5  c.c.  of  the  saturated  neutralized  potassium  oxalate  solution 
which  was  used  in  the  "formol  titration"  of  ammonia.  Explain 
the  results. 

Titrate  the  acidity  of  20  or  25  c.c.  of  urine  (a)  with  and  (b) 
without  the  addition  of  5  c.c.  of  the  neutralized  oxalate  solution. 
From  the  result  obtained  in  (a),  calculate  the  acidity  of  the  twen- 
ty-four hour  urine  in  c.c.  of  decinormal  acid.  Calculate  also  the 
condition  of  the  phosphates  in  the  urine,  assuming  that  the  acidity 
of  normal  urine  is  first  of  all  due  to  acid  phosphates. 

23.  Determination  of  Chlorids. — STANDARD  SILVER  NITRATE 
SOLUTION. — This  is  prepared  by  dissolving  23.94  g.  silver  nitrate 
per  liter  of  solution  (or  5.99  g.  in  250  c.c.). 

STANDARD  AMMONIUM  SULPHOCYANATE  SOLUTION. — Dissolve 
6  g.  of  the  salt  in  800-900  c.c.  water.  Transfer  10  c.c.  of  the  sil- 
ver solution  to  a  beaker  or  flask ;  add  50  c.c.  water,  5  c.c.  concen- 

103 


trated  nitric  acid,  and  2  c.c.  of  saturated  ferric  ammonium  sul- 
phate solution.  By  means  of  a  buret  titrate  the  acidified  silver 
solution  with  the  sulphocyanate  solution.  On  the  basis  of  the 
result,  dilute  a  part  of  the  sulphocyanate  solution  so  as  to  give 
500  c.c.  (or  a  liter)  of  a  solution,  20  c.c.  of  which  is  exactly  equiv- 
alent to  10  c.c.  of  the  silver  solution. 

Each  c.c.  of  the  silver  solution  corresponds  to  5  mg.  chlorin 
(or  to  8.23  mg.  sodium  chlorid). 

The  chlorin  determination  in  urine  is  carried  out  as  follows : 

Pipet  10  c.c.  of  urine  into  a  100  c.c.  volumetric  flask,  add  50  c.c. 
distilled  water,  5  c.c.  saturated  ferric  alum  solution,  and  5  c.c.  con- 
centrated nitric  acid.  Add  20  c.c.  standard  silver  nitrate  solution, 
fill  up  to  the  mark  with  distilled  water,  and  shake.  Filter  through 
a  dry  filter  into  a  dry  beaker  or  flask.  With  a  clean,  dry  pipet 
transfer  50  c.c.  of  the  filtrate  to  another  beaker,  flask,  or  evap- 
orating dish,  and  titrate  in  the  same  manner  as  when  standardizing 
the  silver  solution. 

Since  the  sulphocyanate  solution  is  half  as  strong  as  the  silver 
solution,  and  since  only  one-half  of  the  surplus  silver  was  taken 
for  titration,  20  minus  the  sulphocyanate  titration  figure  repre- 
sents the  silver  nitrate  which  has  combined  with  the  chlorin  of 
the  urine  to  form  silver  chlorid.  This  figure  multiplied  by  5  or 
by  8.23  gives  the  chlorin  or  sodium  chlorid  (in  milligrams)  present 
in  10  c.c.  of  urine. 

Calculate  the  twenty- four  hour  quantity. 

24.  Indicau. — To  10  c.c.  of  urine  add  2  c.c.  of  copper  sulphate 
solution,   5  c.c.  chloroform,  and  an  equal  volume   (17  c.c.)   of 
strong  hydrochloric  acid.     Close  the  mouth  of  the  tube  with  the 
thumb,  and  cautiously  invert  a  few  times. 

The  amount  of  indican  present  is  proportional  to  the  depth  of 
color  of  the  chloroform  extract.  This  qualitative  test  is  often 
made  roughly  quantitative  by  using  the  color  of  Fehling's  solu- 
tion as  a  standard. 

25.  Metabolism    Experiments. — Weigh    accurately    a    small, 
clean,  dry  flask.     Pipet  into  it  25  c.c.  of  urine  and  weigh  again. 
From  the  data  obtained  calculate  the  specific  gravity  of  the  urine. 

Determine  the  specific  gravity  of  the  same  sample  of  urine 
by  means  of  an  ordinary  clinical  areometer.  Compare  the  results 
obtained,  and  explain  how  to  standardize  a  clinical  areometer. 

105 


Collect  a  full  twenty-four  hour  quantity  of  urine,  and  in  it  de- 
termine the  following :  Volume,  specific  gravity,  acidity,  total 
nitrogen,  urea,  ammonia,  uric  acid,  creatinin,  chlorids,  phosphates, 
sulphates,  ethereal  sulphates.  Test  qualitatively  for  indican. 

For  two  days  eat  no  meat,  fish,  eggs,  milk,  cheese,  peas,  or 
beans,  and  only  a  little  bread.  Eat  much  butter,  potatoes,  vege- 
tables, starch  preparations,  fruit,  and  candy.  Repeat  all  the  de- 
terminations with  the  second  twenty-four  hour  quantity. 

For  two  days  eat  all  the  meat  products  you  can,  and  collect  the 
two  twenty-four  hour  quantities  of  urine.  In  the  second  twenty- 
four  hour  quantity  determine  all  the  factors  enumerated  above. 
Tabulate  and  compare  the  results  obtained  in  the  three  series  of 
analyses. 

Take  15  g.  of  sodium  bicarbonate  in  divided  doses,  collect  the 
twenty-four  hour  urine,  and  estimate  the  ammonia  and  acidity. 
Compare  with  the  normal. 

Two  days  later,  beginning  in  the  morning,  take  5  g.  ammonium 
chlorid  in  the  course  of  the  day.  Determine  the  ammonia  and 
acidity. 

Eat  much  sweetbread,  kidney,  or  liver,  for  one  day ;  collect  the 
urine  for  the  whole  twenty-four  hours,  and  estimate  the  uric  acid. 
Compare  with  the  uric  acid  figures  previously  obtained.  Explain. 


107 


ft.it- 


PART  VH 
BLOOD 

1.  Hemoglobin   Crystals. — Place   a   drop   of   defibrinated   rat 
blood  on  a  slide,  add  a  drop  or  two  of  water,  mix,  and  cover  with 
a  cover-glass.     Sketch  the  crystals  which  separate  after  a  few 
minutes. 

2.  Hemoglobin  (Reduced  Hemoglobin). — Add  to  dilute  blood 
a  few  drops  of  strong  ammonium  sulphid,  or  one  or  two  drops 
of  freshly  prepared  Stokes'  reagent. 

Examine  spectroscopically. 

Stokes'  reagent  is  a  2  per  cent,  solution  of  ferrous  ammonium 
sulphate  in  3  per  cent,  tartaric  acid,  to  which  is  added  ammonia 
until  a  clear  solution  is  obtained.  The  ammonia  should  be  added 
only  to  the  amount  of  reagent  immediately  needed. 

Shake  the  solution  of  hemoglobin  with  air,  and  note  the  rapid 
change  to  oxyhemoglobin.  Change  the  same  solution  of  oxy hemo- 
globin to  hemoglobin,  and  reverse  two  or  three  times,  and  note 
the  facility  with  which  hemoglobin  takes  up  and  loses  oxygen. 

3.  Carbon  monoxid  hemoglobin. — Pass  a  current  of  illuminat- 
ing gas  through  a  dilute  oxyhemoglobin  solution  for  a  minute, 
and  filter.    Note  the  change  of  color.    Try  the  effect  on  the  solu- 
tion of  (i)  ammonium  sulphid,  (2)   Stokes'  reagent,  (3)  potas- 
sium ferrocyanid,  (4)  shaking  with  air.    Note  the  stability  of  the 
compound. 

Examine  spectroscopically. 

4.  Methemoglobin. — Add  to  dilute  defibrinated  blood  (1:15) 
two  drops  of  a  freshly  prepared  solution  of  sodium  nitrite.    Note 
the  change.    What  is  the  effect  produced  by  the  addition  of  reduc- 
ing agents  ? 

109 


5.  Hematin.— Hemolyze  a  small  quantity  of  blood  and   add 
dilute  hydrochloric  acid  cautiously  till  a  precipitate  occurs. 

Acidify  strongly  with  hydrochloric  acid.  Note  color  (acid 
hematin).  Then  add  sodium  hydrate  till  strongly  alkaline.  Note 
color  (alkaline  hematin).  To  the  alkaline  solution  add  a  few 
drops  ammonium  sulphid  and  warm  gently.  Note  color  (reduced 
hematin  or  hemochromogen). 

6.  Hemin  Crystals — Teishmann's  Test. — Place  a  bit  of  pow- 
dered dried  blood  on  a  glass  slide,  add  a  minute  crystal  of  sodium 
iodid  and  two  drops  of  glacial  acetic  acid.     Cover  with  a  cover- 
glass  and  warm  gently  over  a  flame  until  bubbles  appear.     De- 
scribe the  crystals  which  separate. 

7.  Sugar  in  Blood. — Mix  25  c.c.  of  fresh  blood  with  150  c.c. 
water  and  heat  to  boiling.     Add  10  c.c.  "colloidal  iron"  solution, 
shake,  and  filter.     Concentrate  the  clear  filtrate  to  about  25  c.c. 
Test  for  sugar. 

8.  Fibrinogen. — Allow  about  6-8  volumes  fresh  blood  to  run 
from  the  animal  into  I  volume  of  a  i  per  cent,  potassium  oxalate 
solution  (why?).    Allow  to  stand  over  night  in  the  cold  room,  and 
siphon  off  the  clear  plasma.    With  the  solution  so  obtained  make 
the  following  experiments : 

Dilute  10  c.c.  with  20  c.c.  of  distilled  water  and  divide  into  3 
equal  portions.  To  one  add  a  little  dilute  (i  per  cent.)  calcium 
chlorid  solution.  To  the  second  add  a  few  drops  of  blood  serum 
(why?).  Place  the  three  tubes  in  a  beaker  of  water  heated  to 
40°  and  observe  the  time  of  clotting. 


in 


PART  vm 

MILK 

Determine  the  specific  gravity  as  in  the  case  of  urine. 

1.  Determination  of  Total  Nitrogen. — Transfer  25  c.c.  milk  to 
a  100  c.c.  volumetric  flask.    Fill  to  the  mark  with  water,  mix,  and 
determine  the  total  nitrogen  in  i  c.c.    Calculate  the  total  protein 
content  of  the  milk  by  multiplying  its  nitrogen  with  the  fac- 
tor 6.25. 

2.  Determination  of  Casein. — Transfer  50  c.c.  of  the  diluted 
milk  to  another  100  c.c.  flask,  and  carefully  precipitate  the  casein 
by  the  addition  of  dilute  acetic  acid  and  gentle  shaking.     Make 
up  to  volume  (100  c.c.)  with  water.    Centrifuge  a  portion,  and  de- 
termine the  nitrogen  in  5  c.c.  of  the  clear  liquid. 

Calculate  the  total  nitrogen  (and  protein),  making  due  allow- 
ance for  the  dilutions,  and  subtract  from  the  total  protein  found 
in  the  preceding  experiment.  The  difference  represents  casein. 

3.  Determination  of  Lactose. — Precipitate  the  proteins,  etc.,  by 
acid  mercuric  nitrate.     This  reagent  is  made  by  dissolving  mer- 
cury in  double  its  weight  of  concentrated  nitric  acid  and  diluting 
with  an  equal  volume  of  water. 

To  50  c.c.  of  milk  in  a  100  c.c.  measuring  flask  add  I  c.c.  of  the 
nitrate  solution,  fill  up  to  the  100  c.c.  mark  with  water,  and  agi- 
tate. Filter  through  a  dry  filter,  and  determine  the  sugar  in  the 
filtrate  by  means  of  the  polariscope.  The  result  will  be  a  trifle  low 
(about  2  per  cent.)  on  account  of  the  volume  occupied  by  the 
curd. 

In  the  case  of  mothers'  milk  it  is  often  inconvenient  to  try  to 
secure  50  c.c.  In  such  cases  10  c.c.  and  a  25  c.c.  volumetric  flask 
may  be  used ;  count  the  drops  necessary  to  give  I  c.c.  of  the  ni- 
trate solution,  and  add  one-fifth  of  that  number  of  drops  (i.  e., 
.2  c.c.)  ;  then  fill  up  to  the  mark,  filter,  and  polarize. 


4.  Determination  of  Fat. — Measure  out  17.6  c.c.  of  thoroughly 
mixed  milk  into  a  Babcock  flask.  Add  17  c.c.  sulphuric  acid  (sp. 
gr.  1.82)  and  mix  thoroughly,  with  gentle  turning  and  shaking, 
until  all  the  precipitated  proteins  have  dissolved.  Rotate  in  the 
centrifuge  for  3  minutes.  Add  hot  water  up  to  the  beginning 
of  the  graduations  in  the  neck  of  the  flask,  and  rotate  for  I  min- 
ute. The  graduations  read  in  per  cent,  of  fat. 


PART  IX 
BONE 

Weigh  a  piece  of  clean,  raw  bone  on  the  laboratory  scales.  Im- 
merse in  about  10  times  its  weight  of  10  per  cent,  hydrochloric  acid 
in  a  flask.  If  any  gas  is  evolved,  determine  what  it  is. 

After  48  hours  dilute  the  volume  of  the  solution  and  what  re- 
mains of  the  bone  to  a  definite  volume  in  a  cylinder.  Mix  so  as 
to  get  the  solution  uniform  in  composition. 

Pipet  out  25  c.c.  of  the  solution,  neutralize  with  sodic  hydrate, 
using  congo  red  paper  as  indicator,  and  determine  the  phosphates. 
Repeat. 

Calculate  the  tricalcic  phosphate  corresponding  to  the  phos- 
phoric acid  found. 

Taking  other  portions  of  the  original  solution,  demonstrate  ex- 
perimentally that  all  the  calcium  in  the  bone  can  not  be  precipi- 
tated together  with  the  phosphoric  acid  present. 

In  what  form  is  this  excess  of  calcium  present  in  bone? 

Examine  the  insoluble  substance  left  in  the  hydrochloric  acid 
solution.  What  is  the  substance?  Prepare  a  "gelatin"  solution 
from  it. 


117 


PART  X 
BILE 

1.  Character  of  Bile. — Determine  the  specific  gravity,  taste, 
odor,  color,  consistency,  reaction,  of  the  bile  supplied. 

Test  for  coagulable  protein. 

2.  Bile  Salts. — Mix  250  c.c.  ox-bile  with  one-fourth  its  volume 
of  bone-black,  and  evaporate  nearly  to  dryness  on  the  water-bath. 
Cool,  transfer  the  residue  to  a  flask,  and  extract  with  200  c.c.  of 
alcohol  over  night.     Filter,  and  evaporate  the  filtrate  to  dryness 
on  the  water-bath.    Dissolve  the  residue  in  absolute  alcohol,  and 
filter  into  a  dry  flask.    Add  anhydrous  ether  till  permanent  cloudi- 
ness develops.    Place  in  the  cold  room  to  crystallize.    Filter.    De- 
scribe the  crystals. 

3.  Pettenkofer's  Test  for  Bile  Salts. — Mix  a  little  bile  with  2 
or  3  drops  of  10  per  cent,  solution  of  cane  sugar.    Place  in  a  test 
tube  some  concentrated  sulphuric  acid.    Incline  the  tube  contain- 
ing the  sulphuric  acid,  and  pour  the  bile  solution  slowly  down 
the  side  of  the  tube  so  that  it  forms  a  layer  above  the  sulphuric 
acid. 

4.  Grmelin's  Test  for  Bile  Pigments. — Put  5  c.c.  of  nitric  acid, 
containing  some  nitrous  acid,  in  a  test  tube,  and  introduce  on  top 
of  it   (pipet)   about  5  c.c.  of  diluted  bile.     Note  what  occurs. 
Study  the  delicacy  of  the  reaction  with  very  dilute  solution  of 
bile. 

5.  Test  for  Bile  in  Urine. — The  presence  of  bile  in  human  urine 
is  usually  indicated  by  its  color  and  the  color  of  the  foam.     In 
making  the  nitric  acid  test  for  albumin  the  presence  of  bile  is 
also  revealed  by  a  series  of  colored  rings  (green,  blue,  violet,  red, 
and  yellowish-red). 

119 


A  similar  series  of  colors  is  occasionally  obtained  from  urines 
which  have  been  preserved  with  thymol.  This  is  one  of  the  objec- 
tions to  this  otherwise  excellent  preservative. 

To  10  c.c.  of  urine  add  a  few  drops  of  calcium  chlorid  solution 
and  a  few  drops  of  10  per  cent,  sodic  hydrate.  Filter.  Remove 
the  filter  paper  from  the  funnel,  open  it,  and  drop  I  or  2  drops 
of  concentrated  nitric  acid  on  the  sediment.  In  the  presence  of 
(human)  bile  the  usual,  characteristic  series  of  colored  rings  is 
obtained. 


121 


SUPPLEMENT 


- 


TJKINE 

Qualitative  Test  for  Acetone  in  Urine. — Clinicians  seldom  dif- 
ferentiate between  acetone  and  diacetic  acid,  and  the  "acetone 
tests"  which  they  use  are  tests  for  diacetic  acid  rather  than  for 
acetone.  (See  p.  133.) 

In  the  qualitative  test  for  acetone,  as  for  its  quantitative  de- 
termination, the  acetone  is  first  removed  from  the  urine  by  means 
of  an  air  current,  just  as  in  corresponding  determinations  (and 
tests)  for  ammonia. 

In  the  large  test  tube  used  for  the  colorimetric  determination 
of  ammonia  place  first  5  c.c.  of  urine  and  1-2  drops  dilute  acid 
(HC1  or  H2SO4).  Then  insert  the  rubber  stopper  carrying  the 
absorption  tube,  etc.,  place  the  test  tube  in  a  beaker  of  lukewarm 
water  (35-40°  C),  and  aspirate  the  volatile  acetone  by  means  of 
a  moderately  rapid  air  current  into  a  test  tube  containing  5  c.c. 
distilled  water  and  5  c.c.  Scott- Wilson  reagent.  If  acetone  is  pres- 
ent, even  if  only  in  minute  traces,  the  solution  becomes  turbid. 
If  the  amount  of  acetone  obtained  is  extremely  small  the  turbidity 
may  not  appear  for  5-10  minutes. 

The  Scott-Wilson  reagent  for  acetone,  which  is  used  for  qualita- 
tive tests  as  well  as  for  quantitative  determinations,  is  most  con- 
veniently prepared  as  follows: 

To  10  g.  of  mercuric  cyanid  dissolved  in  600  c.c.  of  water  add  a 
cooled  solution  of  180  g.  of  sodium  hydroxid  in  600  c.c.  of  water. 
Transfer  this  mixture  to  a  heavy  walled  glass  jar,  and  to  it  add  2.9 
g.  of  silver  nitrate  dissolved  in  400  c.c.  of  water.  The  silver  solution 
should  be  added  in  a  slow  stream,  and  the  addition  must  be  accom- 
panied by  constant  and  exceedingly  vigorous  stirring  with  a  heavy 
glass  rod.  If  properly  made,  the  silver  dissolves  completely,  giving 
a  clear  solution  which  is  at  once  available  for  use.  If  the  solution 
is  turbid,  it  should  be  set  aside  to  settle  for  three  or  four  days  and 
the  clear  supernatant  liquid  removed  by  means  of  a  siphon. 

In  the  clear  reagent  a  new  sediment  gradually  forms,  so  that  the 
solution  deteriorates  slowly  and  after  a  few  months  is  not  service- 
able for  quantitative  work,  though  still  good  for  qualitative  tests. 

125 


Titration  of  Acetone  and  Preparation  of  Standard  Acetone  Solu- 
tions.— Standard  solutions  of  iodin,  sodium  thiosulphate,  and  potas- 
sium permanganate  are  needed  in  this  work,  the  latter  being  used 
only  as  a  basis  for  making  the  other  two  accurate.  The  .05  N  per- 
manganate solution  used  for  the  titration  of  uric  acid  (see  p.  91) 
may  be  used  for  this  purpose. 

lodiru — Weigh  roughly  10-12  g.  of  potassium  iodid  in  a  beaker  and 
add  50  c.c.  of  water.  Weigh  out  6.4  g.  iodin  in  a  small  beaker  cov- 
ered with  a  watch  glass  and  add  this  to  the  potassium  iodid  solution. 
Stir  until  the  iodin  is  dissolved  and  then  transfer  the  resulting  solu- 
tion to  a  500  c.c.  volumetric  flask.  Dilute  to  the  mark  with  water 
and  mix. 

Sodium  Thiosulphate.— Weigh  out  24.85  g.  of  the  salt  (Na2S2O3  + 
5H2O),  dissolve  in  water,  transfer  to  a  500  c.c.  volumetric  flask,  fill 
to  the  mark  with  water,  and  mix. 

The  two  solutions  thus  prepared  should  be  approximately  tenth 
normal.  Their  relative  values  are  determined  by  titration  as  follows : 

Pipet  20  c.c.  of  the  iodin  solution  into  a  flask  (capacity  500-600 
c.c.)  and  add  about  100  c.c.  water.  From  a  buret,  run  in  the  thio- 
sulphate solution  until  the  reddish-brown  iodin  color  has  faded  to  a 
faint  straw  yellow.  Now  add  a  few  drops  of  starch  paste  (see  p.  57) 
and  continue  the  titration  till  the  blue  iodid  of  starch  color  disap- 
pears. The  end  point  of  this  titration  is  very  sharp. 

The  value  of  the  thiosulphate  solution  is  now  determined  as  fol- 
lows: 

Weigh  roughly  2  g.  potassium  iodid,  transfer  to  a  flask,  and  dis- 
solve in  about  150  c.c.  water.  Add  5  c.c.  diluted  hydrochloric  acid 
(1-5)  and  50  c.c.  .05  N  potassium  permanganate  solution. 

The  permanganate  sets  free  an  equivalent  quantity  of  iodin  accord- 
ing to  the  following  equation : 

2KMnO4  -f  loKI  -f  i6HCl  =  I2KC1  +  2MnQ2  +  8H2O  +  sI2 

The  iodin  thus  liberated  is  then  titrated  with  the  sodium  thiosul- 
phate solution  in  the  same  manner  as  the  original  iodin  solution. 

From  the  relative  values  of  the  iodin,  the  thiosulphate,  and  the  per- 
manganate solutions,  the  exact  values  of  the  first  two  (in  terms  of 
tenth  normal  solutions)  are  calculated. 

Standard  Stock  Solution  of  Acetone.— Add  about  i  c.c.  of  pure 
acetone  (from  the  bisulphite  compound)  to  water  in  a  one  liter  volu- 
metric flask,  dilute  to  the  mark,  and  mix.  The  titration  of  acetone 
with  iodin  is  based  on  the  fact  that  in  alkaline  solutions  the  acetone 
is  converted  into  iodoform.  Several  reactions  are  involved  in  this 
process: 

127 


lodin  is  converted  into  hypo-iodite, 

1.  I2  +  2KOH  =  KOI  +  KI  +  H2O. 
Hypo-iodite  is  then  slowly  converted  into   (useless)   iodate, 

2.  3KOI  =  KIO3  +  2KI. 

The  hypo-iodite  converts  acetone  into  iodoform  and  acetic  acid, 

3.  3KOI  +  CH3COCH3  =  CH3  COCI3  +  3KOH. 

4.  CH,  COCI3  +  KOH  =  CH3COOK  +  CHI3. 

On  acidifying,  after  the  iodoform  has  been  formed,  the  surplus 
iodin  present  as  hypo-iodite  (and  iodate)  is  set  free,  and  can  then  be 
titrated  with  the  standard  sodium  thiosulphate  solution  as  described 
above. 

Each  molecule  of  acetone  uses  up  three  molecules  of  hypo-iodite, 
and  as  each  molecule  of  the  latter  is  formed  at  the  expense  of  two 
atoms  of  iodin,  six  atoms  of  iodin  correspond  to  one  molecule  of 
acetone.  One  c.c.  of  .1  N  iodin  solution  corresponds  therefore  to 
.968  mg.  acetone.  Because  of  the  iodate  formation  a  considerable 
excess  must  be  added. 

The  titration  of  the  acetone  solution  is  carried  out  as  follows : 

Transfer  25  c.c.  of  the  stock  acetone  solution  to  a  flask,  add  150- 
200  c.c.  water,  then  50  c.c.  of  the  standardized  iodin  solution,  and  10 
c.c.  strong  sodic  hydrate  (40  per  cent.).  Let  stand  with  occasional 
shaking  for  5  minutes.  Add  18  c.c.  concentrated  hydrochloric  acid, 
and  titrate  the  liberated  excess  of  iodin  with  the  standard  thiosul- 
phate solution. 

If  the  standard  solutions  are  exactly  tenth  normal,  subtract  the 
volume  of  thiosulphate  employed  from  the  volume  of  iodin  solution 
taken,  and  multiply  the  remainder  (in  c.c.)  with  .968  to  obtain  the 
acetone  content  (in  mg.). 

Calculate  the  acetone  content  of  the  stock  solution  (in  mg.  per 
c.c.).  Transfer  to  a  distilling  flask  as  much  of  it  as  contains  exactly 
50  mg.  of  acetone.  Add  water  enough  to  make  a  volume  of  500-600 
c.c.,  and  distill  with  vigorous  cooling  of  the  condenser.  The  receiver 
should  be  a  large  flask  (750-1000  c.c.)  containing  about  250  c.c. 
approximately  normal  sulphuric  acid.  Boil  for  20-30  minutes,  or 
until  at  least  150  c.c.  of  distillate  has  gone  over.  Transfer  this  dis- 
tillate to  a  volumetric  (liter)  flask  and  dilute  to  the  mark  with, 
water.  Ten  c.c.  of  the  acetone  solution  so  obtained  contains  .5  mg. 
acetone.  This  solution,  as  well  as  the  original  stock  solution,  should 
be  kept  in  a  well  stoppered  bottle.  The  sulphuric  acid  present  in  the 
dilute  standard  acetone  solution  is  added  to  prevent  polymerization. 

Preparation  of  Standard  Acetone  Solutions  from  the  Acetone  Bi- 
sulphite Compound. — A  standard  acetone  solution  can  be  prepared 
without  distillations  from  the  "acetone  sulphite"  used  in  photography 
as  follows : 

129 


Transfer  2.5  g.  of  the  powder  to  a  volumetric  (1,000  c.c.)  flask  by 
means  of  a  little  water  (50  c.c.),  and  fill  up  to  the  mark  with  dilute 
(i  in  5)  hydrochloric  acid.  Transfer  25  c.c.  of  the  solution  to  a 
flask.  Add  20  c.c.  tenth  normal  iodin,  let  stand  for  five  minutes,  and 
titrate  the  surplus  iodin  with  tenth  normal  thiosulphate  solution.  This 
titration  gives  the  SO2  or  the  sodium  bisulphite  content. 

To  another  25  c.c.  of  the  acetone  solution  add  50  c.c.  tenth  normal 
iodin,  let  stand  five  minutes,  then  add  10  c.c.  strong  sodic  hydrate, 
followed  after  five  minutes  by  18  c.c.  concentrated  hydrochloric  acid. 
Titrate  the  liberated  iodin  with  thiosulphate.  From  the  50  c.c.  of 
iodin  taken  subtract  (a)  the  figure  of  the  last  thiosulphate  titration 
and  (b)  the  iodin  corresponding  to  the  SO2.  The  remainder  cor- 
responds to  the  acetone.  From  the  standardized  stock  solution  pre- 
pare the  more  dilute  standard  solution  (5  c.c.  or  10  c.c.  of  which 
should  contain  exactly  half  a  mg.  of  acetone). 

If  the  "acetone  sulphite"  is  not  available,  acetone  sodium  bisulphite 
is  easily  prepared  by  slowly  adding  (with  stirring)  two-thirds  volume 
of  ordinary  acetone  to  one  volume  (100  or  200  c.c.)  of  saturated 
sodium  bisulphite  solution  (freshly  prepared  and  filtered).  The  pre- 
cipitate should  be  freed  as  completely  as  possible  from  the  mother 
liquor  by  filtering  on  a  Buchner  funnel  with  suction.  Then  wash 
rapidly  two  or  three  times  with  alcohol.  Let  dry  in  the  open  air 
for  two  or  three  days.  Sieve  to  make  the  preparation  uniform,  and 
preserve  in  glass  stoppered  vessel. 

Quantitative  Determination  of  Acetone  in  Urine. — To  about  i 
c.c.  of  10  per  cent,  sulphuric  acid  in  a  large  test  tube  add  enough 
urine  (.5  to  5  c.c.)  to  give  about  .5  mg.  of  free  acetone  (.3--7 
mg.).  Connect  the  test  tube,  as  in  ammonia  determinations,  with 
a  second  test  tube  containing  10  c.c.  of  fresh  approximately  2 
per  cent,  sodium  bisulphite  solution.  Warm  the  first  test  tube  to 
35-40°  C,  as  in  the  qualitative  test  for  acetone  and  aspirate  the 
acetone  into  the  bisulphite  solution  by  means  of  a  moderate  air 
current  (time  about  10  minutes).  Transfer  the  sulphite-acetone 
solution  to  a  100  c.c.  volumetric  flask  together  with  distilled  water 
enough  to  make  50-60  c.c.  To  each  of  two  other  100  c.c.  flasks 
add  10  c.c.  of  the  standard  acetone  solution  containing  .5  mg. 
acetone,  add  10  c.c.  of  the  2  per  cent,  bisulphite  solution,  and  dilute 
with  distilled  water  to  50-60  c.c. 

To  each  of  the  three  flasks  add  15  c.c.  (clear)  Scott- Wilson  re- 
agent, and  immediately  (before  turbidity  formation)  dilute  with 
distilled  water  to  the  mark,  mix,  and  let  stand  for  12-15  minutes. 
Read  the  turbid  contents  of  one  of  the  standard  acetone  suspen- 


sions  against  itself  in  the  Duboscq  colorimeter.  The  best  source 
of  light  for  these  comparisons  is  diffuse  daylight  coming  through 
an  opening  (about  25  cm.  square)  cut  through  the  shade  of  a 
(north  side)  window.  The  colorimeter  metal  screen  must  also  be 
used.  The  instrument  must  be  adjusted  until  20  m.m.  of  the  two 
suspensions  are  equal.  Now  replace  the  contents  of  one  of  the 
colorimeter  cups  with  the  contents  t)f  the  second  standard  sus- 
pension, and  the  other  with  the  unknown  acetone  mercury  sus- 
pension obtained  from  the  urine,  and  make  the  turbidity  compari- 
son in  the  same  manner  as  colorimetric  comparisons — setting  the 
standard  at  20  m.m. 

Twenty  multiplied  by  .5  and  divided  by  the  reading  of  the  un- 
known (in  m.m.)  gives  the  acetone  content  (in  mg.)  of  the  vol- 
ume of  urine  taken  for  the  analysis. 

Qualitative  Test  for  Diacetic  Acid  (in  traces). — To  5  c.c.  of 
urine  in  a  test  tube  add  1-2  c.c.  dilute  acetic  acid  (10  per  cent.) 
and  a  small  crystal  of  sodium  nitroprussid.  Shake  a  few  times 
to  dissolve  the  salt,  then  add  an  excess  of  concentrated  ammonia 
(2-3  c.c.),  and  mix.  A  violet  color  indicates  diacetic  acid. 

Gerhardt's  Ferric  Chlorid  Test  for  Diacetic  Acid.— This  test  is 
useful  for  showing  the  presence  in  urine  of  relatively  large 
amounts  of  diacetic  acid.  It  is  made  as  follows :  To  5  c-c-  °f 
urine  in  a  test  tube  add  ferric  chlorid  solution  (10  per  cent.), 
drop  by  drop.  At  first  a  white  precipitate  of  ferric  phosphate  is 
obtained,  then,  as  the  addition  of  the  reagent  is  continued,  a  dark 
red  color  is  produced  if  diacetic  acid  is  present  (in  more  than 
traces). 

A  number  of  substances  used  as  drugs,  such  as  salicylic  acid, 
phenacetin,  etc.,  give  a  similar  reaction.  If  confusion  due  to 
such  drugs  is  to  be  suspected,  boil  the  deep  red  solution  for  2-3 
minutes.  If  the  color  is  due  to  diacetic  acid,  it  should  disappear 
during  the  boiling  and  not  reappear  on  cooling.  The  disappear- 
ance is  due  to  the  destruction  by  boiling  of  the  unstable  diacetic 
acid. 

Quantitative  Determination  of  Diacetic  Acid  (and  Acetone). — 
Acetone  urines  contain  from  two  or  three  to  nine  or  ten  times 
as  much  aceto-acetic  acid  as  acetone.  In  strictly  fresh  urines  the 
latter  proportions  prevail;  but  the  older  the  urine  the  greater 

133 


becomes  the  relative  proportion  of  acetone,  because  of  the  spon- 
taneous decomposition  of  the  aceto-acetic  acid.  Urines  giving  a 
strong  ferric  chlorid  reaction  usually  contain  more  than  .5  mg.  of 
aceto-acetic  acid  per  cubic  centimeter,  and  must  be  diluted  so  that 
an  appropriate  fraction  of  I  c.c.  (of  the  original  urine)  can  be 
taken  for  a  determination. 

The  amount  of  urine  taken  should  yield  approximately  .5  mg. 
of  acetone  (from  .3  to  .7  mg.).  Transfer  this  amount  of  urine 
to  a  large  test  tube  containing  I  c.c.  of  10  per  cent,  sulphuric 
acid,  and  connect  with  a  second  test  tube  containing  10  c.c.  of 
2  per  cent,  sodium  bisulphite  solution.  Immerse  the  test  tube 
containing  the  urine  in  a  beaker  of  boiling  water  and  the  second 
test  tube  in  cold  water,  then  pass  through  an  extremely  slow  air 
current  for  ten  minutes.  Increase  slightly  the  speed  of  the  air 
current  and  continue  the  aspiration  for  another  five  minutes. 
The  aceto-acetic  acid  plus  acetone  is  thus  transferred,  in  the  form 
of  acetone,  to  the  bisulphite  solution.  Rinse  the  solution  into  a 
100  c.c.  volumetric  flask,  and  determine  the  acetone  exactly  as  in 
the  determination  of  the  preformed  acetone. 

One  mg.  of  acetone  is  equivalent  to  1.8  mg.  of  aceto-acetic  acid. 
From  the  "total  acetone"  of  the  24-hour  quantity  of  urine  is  sub- 
tracted the  total  preformed  acetone,  and  the  remainder  multi- 
plied by  1.8  gives  the  aceto-acetic  acid. 

Determination  of  Beta-oxybutyric  Acid  in  Urine. — The  method 
described  below  was  at  first  thought  by  its  authors  (Folin  and 
Denis)  to  give  strictly  all  the  beta-oxybutyric  acid  present  in 
urine.  But  it  now  appears  that  the  yield  is  only  85-95  per  cent., 
just  as  in  the  original  method  of  Shaffer. 

The  urine  is  diluted  from  10-100  times,  depending  on  how  much 
of  the  substance  is  present.  The  ammonia  content  of  the  urine 
is  the  best  index  as  to  how  much  urine  is  required  to  yield  the 
desired  amount  of  beta-oxybutyric  acid  (1.5-3.5  nig.).  The  fer- 
ric chlorid  test  for  diacetic  acid  is  also  helpful,  but  without  con- 
siderable experience  only  a  preliminary  determination  can  give 
the  desired  information. 

Measure  diluted  urine,  equivalent  to  1.5-3.5  mg.  of  beta-oxy- 
butyric acid,  into  a  500  c.c.  Kjeldahl  flask,  add  a  little  dilute  sul- 
phuric acid  (5  c.c.),  and  water  enough  to  make  a  volume  of  about 
150  c.c.  Boil  the  mixture  gently  for  ten  minutes  (to  drive  off  the 
preformed  acetone  and  the  diacetic  acid),  then  add  to  the  solution 

135 


(with  a  cylinder)  25  c.c.  of  a  solution  containing  i  per  cent  po- 
tassium dichromate  and  35  per  cent,  sulphuric  acid,  and  connect 
the  flask,  in  the  usual  manner,  with  a  condenser  by  means  of  a 
specially  treated  rubber  stopper. 

The  rubber  stopper  should  be  boiled  twice  for  an  hour  in  10  per 
cent,  sodic  hydrate  solution  (or  better,  heated  in  an  autoclave  in  the 
same  solution  for  half  an  hour  at  130-140°  C),  and  then  thoroughly 
washed.  It  is  also  necessary  to  wrap  the  stopper  thoroughly  in  tin 
foil  during  the  distillation,  so  as  to  exclude  the  volatile  sulphur  im- 
purities which  otherwise  are  given  off  and  interfere  with  the  subse- 
quent turbidity  formation. 

Distill  very  slowly,  for  one  and  one-half  hours,  collecting  the 
distillate  (about  100  c.c.)  in  another  500  c.c.  Kjeldahl  flask,  pre- 
viously charged  with  about  100  c.c.  of  water. 

To  the  distillate  add  a  small  amount  sodium  peroxid  (2  g.), 
and  redistill  by  ordinary  rapid  boiling.  Collect  this  final  distil- 
late in  a  100  c.c.  volumetric  flask  (or  cylinder).  About  80  c.c. 
should  be  obtained. 

Dilute  this  distillate  to  the  100  c.c.  mark  with  distilled  water 
and  mix.  Transfer  from  25  to  50  c.c.  into  a  100  c.c.  volumetric 
flask,  and  determine  the  acetone  content  by  the  turbidity  method, 
as  in  the  case  of  the  two  preceding  (acetone)  determinations.  No 
bisulphite  is  used  in  this  case  to  hold  the  acetone,  and  none  should 
therefore  be  added  to  the  standard.  Each  milligram  of  acetone 
obtained  corresponds  to  1.78  mg.  of  beta-oxybutyric  acid. 

Shaffer's  Short  Method  for  the  Determination  of  Beta-oxybutyric 
Acid. — To  50  c.c.  of  urine  add  100  c.c.  of  water,  then  50  c.c.  of 
basic  lead  acetate  solution  (Goulard's  Ext.  U.S. P.),  and  stir.  Add 
50  c.c.  approximately  normal  NaOH  and  stir  again.  Filter.  A 
clear  filtrate  containing  but  traces  of  lead  or  glucose  is  usually 
obtained,  even  though  the  original  urine  contained  considerable 
quantities  of  sugar.  Traces  of  sugar  do  not  interfere  with 
the  determination. 

Introduce  50  c.c.  of  the  filtrate  into  a  500  c.c.  Kjeldahl  flask 
previously  marked  at  the  level  of  100  c.c.  with  a  "glass  pencil." 
Add  25  c.c.  of  water  and  50  c.c.  of  half  concentrated  sulphuric 
acid.  The  latter,  if  freshly  prepared  by  mixing  with  water  ( I :  i), 
must  be  cooled  before  it  is  used. 

Connect  the  Kjeldahl  flask  with  a  dropping  funnel  and  with  a 

137 


condenser.    Distill  off  about  25  c.c.,  collecting  the  distillate  in  an- 
other Kjeldahl  flask. 

The  first  distillate  thus  obtained  contains  the  preformed  acetone, 
as  well  as  the  acetone  derived  from  the  aceto-acetic  acid  of  the  urine. 
By  adding  to  it  5  c.c.  of  strong  alkali  and  redistilling,  for  10  minutes, 
this  acetone  is  obtained,  in  the  second  distillate,  free  from  impurities, 
and  can  be  titrated  with  iodin  and  thiosulphate. 

After  replacing  the  Kjeldahl  flask  used  as  a  receiver  with  an- 
other one,  the  distillation  of  the  urine  filtrate  is  continued,  while 
adding  slowly  (about  15  drops  per  10  seconds)  a  .2  per  cent,  po- 
tassium bichromate  solution. 

During  this  distillation  the  volume  in  the  distilling  flask  should 
be  kept  at  approximately  100  c.c.  (i.  e.,  at  the  level  indicated  by 
the  pencil  mark).  This  is  readily  accomplished  by  regulating  the 
speed  of  the  distillation  so  that  it  just  about  equals  the  speed  with 
which  the  bichromate  solution  is  added.  The  speed  of  the  oxi- 
dation is  much  greater  with  increasing  concentration  of  sulphuric 
acid.  With  too  great  concentration  of  the  acid,  however,  when 
the  volume  approaches  a  level  of  about  70  c.c.,  the  oxybutyric 
acid  is  in  part  converted  into  crotonic  acid,  and  thus  escapes  oxi- 
dation to  acetone. 

The  bichromate  solution  is  added  only  so  fast  as  to  maintain 
a  very  slight  excess ;  the  blue  green  color  should  largely  predomi- 
nate in  the  boiling  mixture.  Occasionally  it  may  be  necessary  to 
interrupt  the  addition  of  bichromate  for  a  few  minutes,  but  the 
volume  in  the  distilling  flask  should  not  be  allowed  to  sink  below 
85  or  90  c.c.  The  addition  of  bichromate  should  be  continued 
until  (at  the  concentration  of  acid  used)  no  more  appears  to  be 
converted  into  the  green  chromium  salt.  From  50  c.c.  to  100  c.c. 
bichromate  solution  (=  .1  g.-.2  g.  K2C2O7)  is  usually  required  for 
each  distillation.  The  addition  (and  distillation)  lasts  20-30  min- 
utes. 

The  distillate  obtained  must  be  redistilled,  after  the  addition 
of  5  c.c.  strong  alkali  and  about  20  c.c.  of  30  per  cent,  hydrogen 
peroxid.  This  final  distillation  need  not  last  more  than  10  min- 
utes. The  distillate  thus  obtained  is  titrated  in  the  usual  manner 
(p.  127)  with  iodin  and  thiosulphate.  The  yield  of  acetone  ob- 
tained is  about  90  per  cent,  of  the  theoretical  amount  when  work- 
ing with  solutions  of  pure  beta-oxybutyric  acid.  A  correction  of 
10  per  cent,  should  therefore  be  added  to  the  results  obtained. 

139 


Slightly  higher  results  (93-94  per  cent.)  may  be  obtained  by 
a  very  slow  addition  of  the  bichromate,  and  a  considerable  pro- 
longation of  the  distillation  period,  but  since  the  theoretical 
amounts  of  acetone  cannot  be  obtained  the  advantage  so  gained 
is  doubtful. 

Colorimetric  Method  for  the  Determination  of  Phenols  in  Urine. 

— The  phosphotungstic  phosphomolybdic  reagent  described  in  con- 
nection with  the  colorimetric  determination  of  uric  acid  (p.  171) 
was  originally  devised  as  a  reagent  for  phenols,  and  is  serviceable 
for  the  determination  of  phenols  in  urinary  filtrates  from  which 
the  uric  acid  has  been  removed. 

Transfer  10  c.c.  of  ordinary,  or  20  of  very  dilute,  urine  to  a 
50  c.c.  volumetric  flask.  Add  acid  silver  lactate  solution  *  (from 
2  to  10  c.c.)  until  no  more  precipitate  is  obtained,  then  add  a- few 
drops  of  colloidal  iron,  and  shake.  Fill  to  the  mark  with  dis- 
tilled water,  shake  again,  and  filter.  By  means  of  this  precipi- 
tation uric  acid  and  traces  of  proteins  are  quantitatively  removed. 
Transfer  25  c.c.  of  the  filtrate  to  a  50  c.c.  volumetric  flask,  and 
to  it  add  a  sufficient  quantity  of  saturated  sodium  chlorid  solu- 
tion (containing  10  c.c.  of  strong  hydrochloric  acid  per  liter)  to 
precipitate  all  the  silver.  Fill  to  the  mark  with  distilled  water 
and  filter. 

To  determine  "free"  (non-conjugated)  phenols,  place  20  c.c. 
of  this  filtrate  in  a  50  c.c.  flask,  and  treat  with  5  c.c.  of  the  phos- 
photungstic phosphomolybdic  acid  reagent  and  15  c.c.  of  sat- 
urated sodium  carbonate  solution.  After  diluting  to  volume  with 
lukewarm  water  (30-35°  C.)  and  allowing  to  stand  for  twenty 
minutes,  read  the  deep  blue  solution  in  a  Duboscq  colorimeter 
against  a  standard  solution  of  phenol. 

To  determine  total  (free  and  conjugated)  phenols,  transfer 
20  c.c.  of  the- same  filtrate  to  a  large  test  tube,  add  ten  drops  of 
concentrated  hydrochloric  acid,  and  cover  the  test  tube  with  a 
small  funnel  Heat  rapidly  to  boiling  over  a  free  flame,  and  then 
place  in  a  boiling  water-bath  (usually  a  tall  beaker)  for  ten  min- 
utes. At  the  end  of  this  time  remove  the  tube,  cool,  and  trans- 
fer the  contents  to  a  100  c.c.  volumetric  flask.  Add  10  c.c.  of  the 
phosphotungstic  phosphomolybdic  reagent  and  25  c.c.  of  sat- 

*  This  solution  consists  of  a  5  per  cent,  silver  lactate  solution  in  5  per 
cent,  lactic  acid. 

141 


urated  sodium  carbonate  solution.  Make  up  to  volume,  shake, 
and  let  stand  for  20  minutes.  Read  against  a  standard  solution 
of  phenol. 

The  standard  is  a  solution  of  pure  phenol  in  .01  N  HC1,  con- 
taining .5  mg.  of  the  former  substance  in  5  c.c.  To  5  c.c.  of 
the  standard  solution  in  a  100  c.c.  flask  add  10  c.c.  of  the  reagent 
and  25  c.c.  of  saturated  sodium  carbonate  solution.  Fill  up  to 
the  mark  with  water  (at  about  30°  C),  and  make  the  color  com- 
parison in  the  usual  manner,  setting  the  standard  at  20  m.m. 
As  phenol  is  an  exceedingly  hygroscopic  substance,  it  is  necessary 
to  standardize  the  solution  by  means  of  the  iodometric  titration. 

This  titration  is  carried  out  as  follows:  Make  a  phenol  solution 
in  .1  N  HC1,  containing  I  mg.  of  crystallized  phenol  per  c.c.  Trans- 
fer 25  c.c.  of  the  phenol  solution  to  a  250  c.c.  flask,  add  50  c.c.  .1  N 
sodic  hydrate,  heat  to  65°  C.,  add  25  c.c.  .1  N  iodin  solution,  stopper 
the  flask,  and  let  stand  at  room  temperature  thirty  to  forty  minutes. 
Add  5  c.c.  of  concentrated  hydrochloric  acid  and  titrate  excess  of 
iodin  with  .1  N  sodium  thiosulphate  solution.  One  c.c.  of  .1  N  iodin 
solution  corresponds  to  1.567  mg.  of  phenol.  On  the  basis  of  the 
results,  dilute  the  phenol  solution  so  that  10  c.c.  contains  I  mg.  of 
phenol. 

Because  of  the  red  precipitate  in  the  solution  it  is  rather  difficult 
to  see  the  end  point  of  the  titration.  For  those  who  have  not  had 
much  experience  it  may  be  advisable  to  dilute  the  solution  to  a  definite 
volume  (after  adding  the  hydrochloric  acid),  then  to  filter,  and  to 
titrate  a  portion  of  the  filtrate  as  recommended  by  Sutton;  with  a 
little  practice,  however,  the  titration  can  be  made  without  this  pro- 
cedure. 

Quantitative  Determination  of  Hippuric  Acid  in  Urine. — In  this 
method  the  hippuric  acid  is  first  hydrolyzed  and  the  resulting  ben- 
zoic  acid  is  extracted  with  chloroform  and  the  chloroform  solu- 
tion is  titrated  with  standard  alcoholic  sodic  hydrate. 

Transfer  100  c.c.  of  urine  to  an  evaporating  dish,  add  10  c.c. 
5  per  cent,  sodic  hydrate  solution,  and  evaporate  to  dryness  on 
the  water-bath.  Rinse  the  residue  into  a  500  c.c.  Kjeldahl  flask 
by  means  of  25  c.c.  of  water  and  25  c.c.  concentrated  nitric  acid. 
Add  .2  g.  copper  nitrate,  a  couple  of  pebbles  to  prevent  bumping, 
and  boil  very  gently  over  a  microburner  for  four  and  one-half 
hours.  During  this  boiling  a  miniature  Hopkins'  condenser  (made 
from  a  large  test  tube)  is  kept  within  the  neck  of  the  boiling 
flask  to  prevent  loss  of  benzoic  acid  which  is  volatile  with  steam. 

143 


After  cooling  rinse  the  condenser  with  25  c.c.  water,  and  trans- 
fer the  contents  of  the  flask  to  a  separatory  funnel  (capacity  500 
c.c.).  Rinse  the  flask  with  25  c.c.  water,  thus  making  the  total 
volume  in  the  separatory  funnel  100  c.c. 

Add  to  this  solution  55  g.  of  ammonium  sulphate,  shake  until 
dissolved,  and  extract  with  neutral  (freshly  washed)  chloroform 
four  times,  using  50,  35,  25,  and  25  c.c.  of  chloroform  respectively. 
Collect  the  chloroform  extracts  in  another  separatory  funnel,  and 
wash  this  by  shaking  with  100  c.c.  saturated  solution  of  pure 
sodium  chlorid,  to  each  liter  of  which  has  been  added  .5  c.c.  con- 
centrated hydrochloric  acid. 

Draw  off  the  chloroform  which  contains  the  benzoic  acid  into 
a  dry  flask,  and  titrate  with  a  dilute  standardized  sodium  alco- 
holate  solution  and  4-5  drops  of  phenolphthalein  as  indicator. 
The  first  distinct  coloration  diffusing  through  the  whole  liquid 
is  taken  as  the  end  point  without  regard  to  subsequent  fad- 
ing. 

The  sodium  ethylate  solution  is  made  by  dissolving  from  1.8  g. 
to  2.3  g.  metallic  sodium  in  absolute  alcohol  and  diluting  to  a  liter 
with  absolute  alcohol.  It  is  standardized  against  chloroform  solu- 
tions of  benzoic  acid. 

One  cubic  centimeter  of  .1  N  alcoholate  corresponds  to  1.22  mg. 
benzoic  acid  or  1.79  mg.  hippuric  acid. 

Turbidity  Method  for  the  Determination  of  Albumin  in  Urine. — 
To  about  75  c.c.  of  water  in  each  of  two  100  c.c.  volumetric  flasks 
add  5  c.c.  of  a  25  per  cent,  solution  of  sulphosalicylic  acid.  To 
one  flask  add  5  c.c.  of  a  standard  protein  solution,  prepared  as 
described  below,  and  containing  10  mg.  of  albumin.  To  the  other 
add  the  albuminous  urine  I  c.c.  at  a  time  (by  means  of  an  Ost- 
wald  pipet)  until  the  turbidity  obtained  seems  to  be  reasonably 
near  that  of  the  standard.  Fill  the  two  flasks  up  to  the  mark 
with  water,  cautiously  inverting  a  few  times  to  secure  mixing. 
The  standard  must  invariably  first  be  read  against  itself  to  secure 
the  adjustment  of  the  colorimeter  (and  of  the  eye).  Then  re- 
place the  contents  of  one  of  the  Duboscq  colorimeter  cups  by 
the  suspension  of  the  unknown,  and  make  the  turbidity  compari- 
son in  the  usual  manner. 

Set  the  standard  containing  10  mg.  of  protein  at  20  mm.  The 
unknown  must  not  read  less  than  10  nor  more  than  30  mm.  Di- 
viding 200  by  the  product  of  the  reading  of  the  unknown  and 

145 


the  number  of  cubic  centimeters  of  urine  taken,  gives  the  albumin 
in  milligrams  per  cubic  centimeter  of  urine. 

It  is  very  important  not  to  shake  the  albuminous  suspensions 
in  the  volumetric  flasks  because  of  the  tendency  of  the  precipitate 
to  agglutinate.  The  preliminary  mixing  must  therefore  be  accom- 
plished by  means  of  a  few  gentle  inversions. 

The  standard  protein  solution  is  prepared  from  fresh  blood  serum 
free  from  hemoglobin.  For  the  preparation  of  this  serum  either 
slaughter  house  or  normal  human  blood  may  be  used. ,  The  so-called 
blood  serum  sold  for  the  preparation  of  bacteriological  culture  media 
should  be  avoided,  as  it  is  usually  several  days  old  and  is  frequently 
partially  decomposed.  The  dried  preparations  of  "blood  albumin" 
listed  by  chemical  dealers  are  also  not  satisfactory  for  the  prepara- 
tion of  standard  solutions.  To  prepare  the  standard,  dilute  25-35  c-c- 
of  serum  with  a  15  per  cent,  solution  of  chemically  pure  sodium 
chlorid  to  about  1500  c.c.  Mix  and  filter.  By  means  of  nitrogen  de- 
terminations ascertain  the  protein  content  of  the  filtrate  ( protein  = 
N  X  6.25)  and  on  the  basis  of  the  figure  obtained,  dilute  the  solution 
with  15  per  cent,  sodium  chlorid  solution  so  that  it  contains  2  mg.  of 
protein  per  cubic  centimeter.  Sodium  chlorid  in  the  concentration 
mentioned  is  fairly  effective  as  a  preservative.  Nevertheless  it  is  best 
to  saturate  the  standard  albumin  solution  with  chloroform  (20  c.c.). 

The  above  method  is  not  applicable  to  urines  which  are  very 
deeply  colored  with  blood  or  bile  pigments.  The  method  is  of 
course  applicable  to  other  albuminous  fluids  than  urine,  as,  for 
example,  exudates,  transudates,  and  the  cerebrospinal  fluid. 

Gravimetric  Method  for  the  Determination  of  Albumin  in  Urine. 
— The  method  is  as  follows:  Pipet  10  c.c.  of  urine  into  an  ordi- 
nary conical  centrifuge  tube,  which  has  been  previously  weighed ; 
add  i  c.c.  of  5  per  cent,  acetic  acid,  and  let  stand  for  fifteen  min- 
utes in  a  beaker  of  boiling  water.  At  the  end  of  this  time  remove 
the  tube  from  the  water-bath  and  centrifuge  for  a  few  minutes. 
Pour  off  the  supernatant  liquid,  stir  up  the  precipitate  in  the  tube 
with  about  10  c.c.  of  boiling  .5  per  cent,  acetic  acid,  and  again 
centrifuge.  Remove  the  supernatant  liquid  from  the  precipitate 
in  the  tube  and  wash  once  more,  this  time  with  50  per  cent,  alco- 
hol. After  centrifuging  and  pouring  off  the  supernatant  alcohol, 
place  the  tube  for  two  hours  in  an  air  bath  at  100-110°,  then  cool 
in  a  desiccator,  and  weigh. 

M7 


McCrudden's  Method  for  the  Determination  of  Calcium  and 
Magnesium  in  Urine  (/.  Biol.  Chem.,  j,  82  and  10,  187). — If  the 
urine  is  alkaline,  make  it  neutral  or  slightly  acid  to  litmus.  Fil- 
ter. Transfer  200  c.c.  of  the  filtered  urine  to  a  small  flask.  Make 
just  alkaline  with  concentrated  ammonium  hydrate  and  then  just 
acid  with  (concentrated)  hydrochloric  acid.  The  cloud  of  phos- 
phates forming  in  alkaline  urine  may  be  used  as  a  guide  in  the 
process  of  acidifying  the  urine.  Coo^  for  a  few  minutes  in  run- 
ning water.  Add  10  drops  of  concentrated  hydrochloric  acid  and 
10  c.c.  of  2.5  per  cent,  oxalic  acid.  Now  add  8  c.c.  of  20  per 
cent,  sodic  acetate  solution,  stopper,  and  shake  vigorously  and 
continuously  for  about  ten  minutes.  Filter  on  a  small  ash  free 
filter  paper  and  wash  free  from  chlorids  with  .5  per  cent,  ammo- 
nium oxalate  solution.  Transfer  the  filter  and  precipitate  to  a 
weighed  platinum  crucible,  dry  over  a  small  flame,  and  then  heat 
in  the  blast  lamp  to  constant  weight,  thus  transforming  the  cal- 
cium oxalate  to  calcium  oxid.  Cool  in  a  desiccator  and  weigh. 

In  the  combined  filtrate  and  washwater  the  magnesium  is  de- 
termined as  follows:  Transfer  the  filtrate  to  a  large  porcelain 
dish,  add  20  c.c.  concentrated  nitric  acid,  and  boil  down  almost  to 
dryness.  When  the  residue  is  nearly  dry  and  no  more  nitrous 
fumes  are  given  off,  add  10  c.c.  concentrated  hydrochloric  acid 
and  again  boil  down  nearly  to  dryness.  Dilute  with  water  to  a 
volume  of  almost  80  c.c.,  and  with  constant  stirring  add  ammonia, 
drop  by  drop,  until  the  mixture  is  alkaline  to  litmus  paper.  Then 
add  25  c.c.  dilute  ammonia  (sp.  gr.  .96)  slowly  and  with  stirring, 
and  set  aside  over  night  in  a  cool  place.  Filter  on  a  small  filter 
paper,  and  wash  the  precipitate  with  a  dilute  solution  of  alcohol 
and  ammonia  (r  volume  of  alcohol  and  I  volume  dilute  ammonia 
mixed  with  3  volumes  of  water).  Wash  until  the  filtrate  is  free 
from  chlorids.  Dry  the  filter  and  ignite  in  a  weighed  platinum 
crucible.  Cool  and  weigh.  The  residue  is  Mg2P2O7. 

Method  for  the  Determination  of  Sodium  and  Potassium  in  Urine. 

— Transfer  50  c.c.  of  urine  to  a  platinum  dish  (capacity  about  250 
c.c.),  evaporate  to  dryness,  and  then  heat  the  residue,  at  first 
very  cautiously,  over  a  radial  burner.  Continue  the  heating  at  a 
barely  perceptible  dull  red  heat  for  one  hour.  Cool.  Moisten  the 
residue  with  20  c.c.  distilled  water,  evaporate  to  dryness,  and  heat 
as  before  for  another  hour.  To  the  residue,  which  now  should 
contain  very  little  carbon,  add  50  c.c.  water  and  5-6  drops  con- 

149 


centrated  hydrochloric  acid.  The  mineral  constituents  are  thus 
brought  into  solution.  Add  an  excess  of  saturated  barium  hy- 
droxid  solution  (i.  e.,  to  a  distinctly  alkaline  reaction),  heat  to 
boiling,  and  filter  on  a  Gooch  crucible.  Wash  with  hot  water. 
The  filtrate  should  now  be  substantially  free  from  calcium,  mag- 
nesium, phosphoric  acid,  and  sulphuric  acid,  but  does  contain 
barium  in  addition  to  the  sodium  and  potassium.  Precipitate  the 
barium  by  passing  washed  carbonic  acid  through  the  solution. 
Filter  on  another  Gooch  crucible  and  wash  with  a  little  cold 
water.  Render  the  filtrate  slightly  acid  to  methyl  orange  (one 
drop),  and  evaporate  to  dryness  in  a  previously  weighed  plat- 
inum dish.  Heat  the  residue  very  gradually  and  carefully  to  a 
dull  red  heat  for  10  minutes.  Cool  in  a  desiccator  and  weigh. 
The  increase  in  weight  gives  the  sodium  and  potassium  as  chlorids. 
Dissolve  the  residue  in  a  very  small  quantity  of  water  and 
add  a  few  drops  dilute  hydrochloric  acid.  Then  add  10  per  cent, 
chlorplatinic  acid  solution  (4-5  times  as  much  H2PtCl6  as  the 
combined  weight  of  the  chlorids  present),  and  evaporate  at  me- 
dium temperature,  about  75°  C.,  until  the  residue  looks  dry.  Now 
add  95  per  cent,  alcohol,  filter  on  a  weighed  Gooch  crucible,  and 
wash  several  times  with  95  per  cent,  alcohol.  Dry  at  110°  and 
weigh.  The  potassium  chlorplatinate  thus  obtained  multiplied  by 
the  factor  .3056  gives  the  corresponding  weight  of  potassium 
chlorid.  The  sodium  chlorid  is  then  obtained  by  subtracting  the 
weight  of.  the  potassium  chlorid  from  the  weight  of  the  combined 
chlorids. 

In  connection  with  this  determination  there  are  two  fruitful  sources 
of  error :  contamination  of  the  chlorplatinate  precipitate  with  ammonia 
(which,  as  ammonium  chlorplatinate,  gives  too  high  results  for  potas- 
sium), and  overheating  during  the  ashing  (which  causes  volatiliza- 
tion of  the  sodium  chlorid).  Loss  of  chlorids  through  overheating 
may  be  avoided  by  placing  the  platinum  dish  containing  the  dried 
urine  on  fragments  of  clay  plate,  or  pieces  of  a  broken  evaporating 
dish,  contained  in  a  shallow  iron  dish  (about  20  cm.  in  diameter) 
which  is  heated  by  means  of  a  large  size  radial  burner. 

Indicator  Method  for  the  Determination  of  the  Hydrogein-ion 
Concentration  in  Urine  (Henderson  and  Palmer,  J.  Biol.  Chem., 
J3>  393)- — Tm"s  determination,  though  simple  in  theory,  is  rather 
complicated  in  the  practical  execution  because  of  the  many  stand- 
ard solutions  which  must  be  used  in  each  determination.  A  defi- 


nite  amount  of  a  suitable  indicator  is  added  to  a  definite  volume 
of  urine  (in  250  c.c.  water)  and  to  each  of  a  series  of  solutions 
of 'known  hydrogen-ion  concentration  (in  250  c.c.  water).  By 
matching  of  colors  the  solution  having  approximately  the  same 
hydrogen-ion  concentration  as  the  urine  is  thus  found  by  inspec- 
tion. The  hydrogen-ion  concentration  is  usually  expressed,  in  ac- 
cordance with  the  practice  of  Sorensen,  in  terms  of  its  logarithm 
(Pn),  with  the  negative  sign  left  out. 

The  standard  solutions  employed  by  Henderson  and  Palmer  can 
be  prepared,  perhaps  most  conveniently,  from  standardized  solutions 
of  sodic  hydrate,  phosphoric  acid,  and  acetic  acid.  Half  normal  and 
tenth  normal  sodic  hydrate,  half  normal  and  tenth  normal  phosphoric 
acid  (figured  as  a  dibasic  acid),  and  half  normal  and  tenth  normal 
acetic  acid,  are  the  preliminary  required  stock  solutions.  The  sodic 
hydrate  should  be  standardized  against  accurate  half  normal  hydro- 
chloric acid,  and  should  be  as  free  as  possible  from  carbonates.  It 
must  also  be  free  from  calcium  or  barium. 

With  accurate  half  normal  sodic  hydrate  as  the  starting  point, 
prepare  a  phosphoric  acid  solution  which  is  somewhat  stronger  than 
half  normal,  taking  account  of  only  two  of  the  three  hydrogen  atoms 
in  the  acid  molecule.  Titrate  25  c.c.  of  this  acid  to  which  has  been 
added  10  g.  neutral  sodium  chlorid,  but  no  extra  water.  Use  phenolph- 
thalein  as  indicator.  On  the  basis  of  the  titration  figures,  prepare 
one  or  two  liters  of  half  normal,  and  one  liter  of  tenth  normal  phos- 
phoric acid.  The  acetic  acid  solutions  are  prepared  in  a  similar 
manner. 

SOLUTION  I.  (.1000  N  Na2HPO4  PH  9.27,  used  with  phenolph- 
thalein  as  indicator.) 

To  200  c.c.  .5  N  phosphoric  acid  add  200  c.c.  .5  N  sodic  hydrate, 
and  dilute  to  500  c.c. 

SOLUTION  2.  (.0001  N  NaH2PO4,  .0480  N  Na2HPO4,  PH  8.7,  used 
with  phenolphthalein  as  indicator.) 

To  96.1  c.c.  .5  N  phosphoric  acid  add  96.05  c.c.  .5  N  sodic  hydrate, 
and  dilute  to  500  c.c. 

SOLUTION  3.  (.0001  N  NaH2PO4,  .0120  N  Na2HPO4,  PH  8.0,  used 
with  alizarin  red,  neutral  red,  or  phenolphthalein,  as  indicator.) 

To  120.5  c-c-  -1  N  phosphoric  acid  add  120.25  c.c.  .1  N  sodic  hy- 
drate, and  dilute  to  500  c.c. 

SOLUTION  4.  (.0166  N  NaH2PO4,  .0060  N  Na2HPO4,  PH  7.48, 
used  with  alizarin  red,  or  neutral  red,  as  indicator.) 

To  183.2  c.c.  .5  N  phosphoric  acid  add  174.9  c.c.  .5  N  sodic  hydrate, 
and  dilute  to  500  c.c. 

153 


SOLUTION  5.  (.0010  N  NaH2PO4,  .0060  Na2HPO4,  PH  7.38,  usec 
with  alizarin  red,  or  neutral  red,  as  indicator.) 

To  65  c.c.  .1  N  phosphoric  acid  add  62.5  c.c.  .1  N  sodic  hydrate, 
and  dilute  to  500  c.c. 

SOLUTION  6.  (.0010  N  NaH2PO4,  .0023  N  Na2HPO4,  PH  6.9,  used 
with  alizarin  red,  or  neutral  red,  as  indicator.) 

To  28  c.c.  .1  N  phosphoric  acid  add  25.5  c.c.  .1  N  sodic  hydrate, 
and  dilute  to  500  c.c. 

SOLUTION  7.  (.0009  N  CH3COOH,  .0920  N  CH3COONa,  PH  6.7, 
used  with  alizarin  red  as  indicator.) 

To  50.5  c.c.  .1  N  acetic  acid  add  46  c.c.  .1  N  sodic  hydrate  and 
dilute  to  500  c.c. 

SOLUTION  8.  (.0023  N  CH3COOH,  .0920  N  CH3COONa,  PH  6.3, 
used  with  alizarin  red  as  indicator.) 

To  57.5  c.c.  .1  N  acetic  acid  add  46  c.c.  .1  N  sodic  hydrate  and 
dilute  to  500  c.c. 

SOLUTION  9.  (.0046  N  CH3COOH,  .0920  N  CH3COONa,  PH  6.0, 
used  with  alizarin  red  as  indicator.) 

To  69  c.c.  .1  N  acetic  acid  add  46  c.c.  .1  N  sodic  hydrate  and  dilute 
to  500  c.c. 

SOLUTION  10.  (.0092  N  CH3COOH,  .0920  N  CH3COONa,  PH  5.7, 
used  with  alizarin  red  as  indicator.) 

To  92  c.c.  .1  N  acetic  acid  add  46  c.c.  .1  N  sodic  hydrate  and  dilute 
to  500  c.c. 

SOLUTION  n.  (.023  N  CH3COOH,  .0920  N  CH3COONa,  PH  5.3, 
used  with  alizarin  red,  or  methyl  red,  as  indicator.) 

To  161  c.c.  .1  N  acetic  acid  add  46  c.c.  .1  N  sodic  hydrate  and  dilute 
to  500  c.c. 

SOLUTION  12.  (.0460  N  CH3COOH,  .0920  N  CH3COONa,  PH  4-9, 
used  with  alizarin  red,  or  methyl  red,  as  indicator.) 

To  55.1  c.c.  .5  N  acetic  acid  add  46  c.c.  .1  N  sodic  hydrate  and  dilute 
to  500  c.c. 

SOLUTION  13.  (.0920  N  CH3COOH,  .0920  N  CH3COONa,  PH  4-7> 
used  with  alizarin  red,  or  methyl  red,  as  indicator.) 

To  1 10  .2  c.c.  .5  N  acetic  acid  add  46  c.c.  .1  N  sodic  hydrate  and 
dilute  to  500  c.c. 

The  determination  is  carried  out  as  follows :  Transfer  10  c.c. 
urine  to  a  250  c.c.  Florence  flask.  A  large  number  of  exactly 
similar  flasks  (20-30)  are  indispensable  for  this  determination. 
Transfer  10  c.c.  of  standard  solution  to  each  of  a  series  of  similar 
flasks,  numbering  each  flask  according  to  the  standard  solution 
it  contains,  then  fill  all  the  flasks  to  the  neck  with  distilled  water, 
and  add  the  indicator  (7.5  mg.  alizarin  red).  Determine  by  in- 

155 


spection  which  standard  solution  most  nearly  matches  the  urine. 

If  the  result  obtained. in  the  determination  with  alizarin  red 
indicates  an  acidity  greater  than  5.3  (Pn),  the  determination 
should  be  repeated,  with  .15  c.c.  saturated  solution  of  methyl  red 
(in  50  per  cent,  alcohol),  as  indicator. 

With  urines  having  an  acidity  of  5.3-6.7  (Pn)  repeat  the  de- 
termination, as  with  alizarin  red,  but  employing  neutral  red  (15 
mg.)  as  indicator. 

With  urines  which  are  alkaline  to  phenolphthalein  use  phenol- 
phthalein  as  indicator. 

In  the  case  of  albuminous  urines  the  original  determination 
should  be  made  with  methyl  red,  instead  of  alizarin  red,  as  in- 
dicator. 


157 


BLOOD 

Determination  of  Non-protein  Nitrogen  in  Blood  (/.  Biol.  Chem., 
ii,  527). — To  a  50  c.c.  volumetric  flask  add  25-35  c.c.  pure  "ace- 
tone free"  methyl  alcohol.  Then  add  with  a  pipet  and  with  con- 
stant shaking  5  c.c.  of  blood.  Fill  to  the  mark  with  methyl  alcohol, 
shake,  and  let  stand  for  two  hours.  Filter  on  a  dry  filter  paper ; 
to  the  filtrate  add  two  or  three  drops  of  saturated  alcoholic  solu- 
tion of  zinc  chlorid  (the  latter  must  be  free  from  ammonia). 
After  standing  for  a  few  minutes  filter  again ;  a  clear  and  per- 
fectly colorless  filtrate  should  be  obtained. 

Transfer  10  c.c.  of  the  alcoholic  filtrate  to  a  large  Jena  test 
tube  of  the  kind  used  in  urine  analysis.  Add  one  drop  of  sul- 
phuric acid,  one  of  kerosene,  and  a  pebble.  Drive  off  the  methyl 
alcohol  by  immersing  the  test  tube  in  a  beaker  ot  boiling  water 
for  five  to  ten  minutes.  When  the  alcohol  is  removed,  add  I  c.c. 
of  concentrated  sulphuric  acid,  a  gram  of  potassium  sulphate, 
and  a  drop  of  copper  sulphate  solution.  Boil  the  mixture,  cool, 
and  dilute  as  in  the  analysis  of  urine  (see  p  85). 

From  this  digestion  mixture  remove  the  ammonia  in  the  usual 
manner,  and  collect  it,  not  in  a  measuring  flask  (as  in  urine  anal- 
ysis), but  in  a  second  large  test  tube  previously  charged  with  I 
c.c.  of  .1  N  acid  and  2  to  3  c.c.  of  water.  The  reason  for  this 
variation  is  that  I  c.c.  of  blood  contains  usually  not  over  .2-.3  mg. 
of  non-protein  nitrogen.  The  final  Nesslerized  solution,  therefore, 
cannot  be  diluted  to  100  c.c.,  and  smaller  volumetric  flasks  cannot 
be  used  as  receivers  during  the  air  current  treatment  because 
of  spattering.  Large  test  tubes  are  therefore  used  as  receivers, 
and  the  ammonia  is  Nesslerized  in  these  before  the  liquids  are 
transferred  to  measuring  flasks.  Ordinarily  the  colored  solutions 
obtained  are  transferred  to  10  c.c.  flasks,  and  are  then  found  to 
have  a  depth  of  color  which  permits  of  a  sure  and  accurate  read- 
ing in  the  colorimeter. 

In  all  ordinary  cases  use  7  to  8  c.c.  of  diluted  Nessler's  reagent 
(dilution  1:5)  for  the  production  of  the  color.  If  much  ammonia 

159 


is  present,  so  that  the  resulting  colored  solution  must  be  diluted 
to  50  c.c.,  correspondingly  larger  amounts  of  Nessler's  reagent 
should  be  added. 

Make  the  colorimetric  comparison  with  a  standard  of  I  mg. 
ammonia  nitrogen,  obtained  from  ammonium  sulphate  and  Ness- 
lerized  as  in  urine  analysis  (in  100  c.c.  flasks). 

Calculate  the  results  to  milligrams  of  non-protein  nitrogen  per 
100  c.c.  of  blood. 

CALCULATION  :  Multiply  20  (the  colorimeter  standard  in  m.m.) 
with  10  (or  any  other  volume  in  c.c.  to  which  the  Nesslerized  un- 
known has  been  diluted),  and  divide  by  R  (the  reading  of  the 
unknown  in  m.m.).  The  result  will  be  found  to  correspond  to 
the  non-protein  nitrogen  in  mg.  per  100  c.c.  of  blood. 

Before  cancellation  the  formula  is: 

20       10     100 

— X X —  mg.  per  100  c.c. 

R       100       i 
(i)   (2)     (3) 

The  first  term  (i)  represents  the  colorimetric  comparison,  the 
second  term  (2)  the  relative  dilutions  of  the  Nesslerized  solu- 
tions, and  the  third  term  (3)  the  volumes  of  blood  involved. 

Preparation  of  Nessler's  Reagent. — Dissolve  200  g.  of  potassium 
hydroxid  (or  sodium  hydroxid)  in  950  c.c.  of  distilled  water  in  a 
flask,  and  cool  under  running  water.  Transfer  40  c.c.  of  distilled 
water,  55  g.  of  potassium  iodid,  and  100  g.  of  mercuric  iodid  to  a 
large  heavy  beaker  (capacity  1500  c.c.)  or  a  glass  jar.  Immerse  the 
lower  part  of  the  beaker  in  warm  water  (40-50°  C),  and  shake 
gently  until  the  last  trace  of  the  red  iodid  of  mercury  is  dissolved. 
The  solution  occurs  rapidly,  and  is  usually  finished  in  10-15  minutes. 
Remove  from  the  warm  water,  and  pour  the  cooled  hydroxid  solution 
into  the  yellow  iodid  mixture  in  a  slow  but  continuous  stream  and 
with  vigorous  stirring.  The  mixture  should  remain  clear,  or  at  the 
most  slightly  opalescent.  If  a  moderate  sediment  has  formed  or 
should  later  form  in  the  solution,  the  clear  supernatant  liquid  is  still 
good.  .Keep  in  well-stoppered  bottles,  preferably  in  the  dark. 

Determination  of  Urea  in  Blood  (/.  BioL  Chem.,  n,  531). — 
Measure  10  c.c.  of  the  alcoholic  filtrate  used  for  the  determina- 
tion of  the  non-protein  nitrogen  into  one  of  the  large  Jena  test 
tubes  in  which  the  decomposition  of  the  urea  is  to  be  made.  Add 

161 


a  drop  of  dilute  acetic  acid,  and  two  or  three  of  kerosene,  and 
close  the  test  tube  by  a  two-hole  rubber  stopper.  (Through  one  of 
the  holes  in  the  stopper  is  passed  a  glass  tube  drawn  out  to  a  capil- 
lary several  inches  long.  The  capillary  end  reaches  nearly  to  the 
bottom  of  the  test  tube.  Through  the  other  hole  passes  a  short 
bent  glass  tube  which  is  connected  with  a  good  water  pump.) 
Place  the  test  tube  in  warm  water,  and  start  the  vacuum  pump. 
In  ten  to  thirty  minutes  the  combined  action  of  the  gentle  heat, 
the  air  current  (through  the  capillary),  and  the  vacuum,  removes 
all  the  alcohol.  Remove  the  rubber  stopper,  breaking  off  the 
capillary  tube  by  bending  it  against  the  sides  of  the  test  tube.  Add 
2  c.c.  of  25  per  cent,  acetic  acid,  a  temperature  indicator,  a  pebble, 
and  7  g.  of  dry  potassium  acetate;  then  heat  to  153-158°  C.  for 
about  eight  to  ten  minutes,  exactly  as  in  the  urea  determination 
described  for  urine  (see  p.  87). 

Collect  the  ammonia,  set  free  by  the  subsequent  air  current 
treatment,  in  a  large  test  tube,  Nesslerize  (usually  with  only  3  c.c. 
of  the  diluted  reagent),  make  up  to  volume  in  a  10  c.c.  volumetric 
flask,  and  make  the  color  comparison,  as  in  the  case.of  the  total 
non-protein  nitrogen,  against  the  same  standard  solution  of  am- 
monium sulphate. 

Urease  Method  for  the  Determination  of  Urea  in  Blood  (Mar- 
shall, J.  Biol.  Chem.,  15,  487). — Prepare  a  dilute  urease  solution 
by  mixing  one  volume  of  the  urease  extract  used  for  the  deter- 
mination of  urea  in  urine  (p.  89)  with  nine  volumes  of  water. 

Blood  seems  to  contain  a  substance  ("auxo-urease")  which 
greatly  accelerates  the  action  of  soy  bean  urease  (about  eight 
times).  ( Neumann :  Block.  Zeitschr.,  Vol.  6p .)  Since  all  urease 
extracts  contain  traces  of  free  ammonia,  the  use  of  unnecessarily 
large  amounts  of  the  ferment  is  to  be  avoided. 

With  a  pipet  containing  a  little  powdered  sodium  citrate  in  the 
tip,  measure  5  c.c.  of  blood  with  gentle  shaking  into  a  25  c.c. 
volumetric  flask  half  full  of  water.  Fill  to  the  mark  with  water, 
using  a  drop  or  two  of  -ether  to  dispel  the  foam,  and  shake  thor- 
oughly. 

Measure  5  c.c.  of  the  diluted  blood  into  a  large  test  tube.  Add 
2  drops  of  kerosene  and  I  c.c.  of  the  diluted  urease  extract. 
Stopper  with  the  aeration  apparatus,  and  let  stand  15  minutes. 
Attach  the  apparatus  to  an  absorption  tube,  the  latter  reaching 
to  the  bottom  of  a  large  test  tube  containing  I  c.c.  tenth  normal 


HC1  and  2  c.c.  of  water.  At  the  end  of  the  15  minutes  pass  the 
air  current  through  the  apparatus  for  a  few  seconds  (to  sweep 
out  any  ammonia  which  exists  as  a  vapor).  Stop  the  air,  open 
the  test  tube,  and  add  about  5  g.  solid  potassium  carbonate.  Close 
the  tube,  and  aerate  in  the  usual  manner  for  10  to  15  minutes. 

To  the  contents  in  the  receiver  add  .5  c.c.  Nessler's  reagent  pre- 
viously diluted  with  2  to  3  volumes  of  water.  Transfer  the  Ness- 
lerized  solution  to  a  10  c.c.  volumetric  flask  (using  less  than  5  c.c. 
of  water  for  washing),  and  fill  up  to  the  mark.  Compare  the 
color  with  that  of  the  standard  ammonium  sulphate  solution  Ness- 
lerized  in  the  usual  manner. 

The  Determination  of  Ammonia  in  Blood  (J.Biol.  Chem.,  11,534). 
— Reasonably  accurate  determinations  of  ammonia  in  blood  are  ob- 
tained with  great  difficulty  because  of  the  decomposition  of  certain 
nitrogenous  components  of  blood  even  at  room  temperatures,  and 
because  the  free  ammonia  actually  present  in  fresh  blood  amounts 
only  to  a  few  hundredths  of  a  milligram  per  100  c.c. 

Transfer  10  c.c.  of  blood  to  a  large  test  tube.  Add  2-3  c.c.  of 
a  solution  containing  10  per  cent,  sodic  carbonate  and  15  per  cent, 
potassium  oxalate.  Then  aspirate  the  liberated  ammonia  by 
means  of  a  rapid  air  current  into  a  test  tube  containing  I  c.c.  of 
water  and  5-6  drops  .1  N  hydrochloric  acid.  Time  20-30  min- 
utes. Nesslerize  this  solution  by  the  gradual  addition  of  not  over 
i  c.c.  diluted  Nessler's  solution  (dilution  1:5).  Transfer  the 
solution  to  a  10  c.c.  volumetric  flask,  and  dilute  to  volume  with 
"ammonia  free"  water. 

"Ammonia  free"  water  is  obtained  from  ordinary  distilled  water 
by  the  addition  of  a  little  bromin  water  and  a  few  drops  of  concen- 
trated sodium  hydroxid. 

The  colorimetric  valuation  of  the  solution  by  means  of  the  Du- 
boscq  colorimeter  cannot  be  accomplished  without  materially  alter- 
ing the  instrument.  An  iris  diaphragm  should  be  attached  to  one 
sliding  platform  of  the  colorimeter,  so  as  to  regulate  the  amount 
of  light  passing  through  on  that  side.  The  hexagonal  prism 
should  be  removed  from  the  opposite  side.  With  these  alterations 
the  Nesslerized  solution  in  a  100  m.m.  polariscope  tube  may  be 
compared  with  .5  or  I  mg.  ammonia  (Nesslerized  and  diluted  to 
100  c.c.).  Place  the  standard  in  the  cup  on  the  side  of  the  iris 

165 


diaphragm,  fill  a  100  m.m.  polariscope  tube  with  the  unknown, 
and  insert  this  on  the  other  side.  Adjust  the  standard  until  the 
two  fields  are  equal. 

Method  for  the  Determination  of  Uric  Acid  in  Human  Blood 

(Compare  J.  Biol.  Chem.,  13,  469  and  20,  629), — Pipet  25  c.c.  of 
distilled  water  into  a  50  c.c.  Erlenmeyer  flask,  and  with  a  pipet 
add  5  c.c.  of  blood.  Rinse  the  latter  pipet  once  or  twice  with  the 
diluted  blood  in  the  flask.  Heat  the  mixture  for  4-5. minutes  by 
immersion  in  a  beaker  of  boiling  water,  rotating  occasionally  so  as 
to  secure  a  uniform  coagulation.  Next  add  i  c.c.  of  an  acid  sodic 
acetate  solution  (made  by  dissolving  I  g.  glacial  acetic  acid  and 
12.5  g.  crystallized  sodic  acetate  in  10  c.c.  of  water),  mix,  and  heat 
a  moment  more  to  complete  the  coagulation.  Remove  the  flask 
from  the  water-bath,  add  4  c.c.  of  diluted  (2.5  per  cent.)  colloidal 
iron  solution  (equal  parts  of  commercial  colloidal  iron  and  distilled 
water),  and  mix  thoroughly.  Filter  through  a  small,  dry,  folded 
filter  into  a  dry  flask.  At  the  end  of  the  filtration  press  the  filter 
paper  containing  the  coagulum  gently  against  the  side  of  the 
funnel  so  as  to  obtain  the  maximum  amount  of  filtrate. 

Transfer  n  c.c.  of  the  blood  filtrate  to  a  centrifuge  tube,  add 
2  c.c.  of  acid  silver  lactate  solution  (silver  lactate  5  per  cent., 
lactic  acid  5  per  cent.),  stir  the  mixture  with  a  small  glass  rod, 
and  rinse  the  rod  with  a  few  drops  of  distilled  water.  Centrifuge 
for  2-3  minutes  and  pour  off  the  supernatant  liquid.  Then  add 
10  c.c.  more  of  the  blood  filtrate  to  the  same  tube,  and  also  an- 
other 2  c.c.  of  the  silver  lactate  solution.  After  stirring  and  rins- 
ing off  the  rod,  centrifuge  again  for  2-3  minutes,  and  pour  off 
the  liquid. 

The  centrifuge  tube  now  contains  the  precipitate  from  21  c.c. 
of  the  blood  filtrate.  Since  the  coagulation  mixture  before  fil- 
tration contained  25  c.c.  of  water,  5  c.c.  of  blood,  4  c.c.  iron  solu- 
tion, and  I  c.c.  of  acetate  solution  (a  volume  of  35  c.c.),  21  c.c. 
of  the  filtrate  represents  3  c.c.  of  blood. 

Add  to  the  precipitate  in  the  centrifuge  tube  about  5  c.c.  of 
water  and  3  drops  magnesia  mixture,  stir  the  precipitate  with  a 
rod,  and  add  ammonia  (1-2  c.c.)  until  the  silver  chlorid  is  com- 
pletely dissolved  and  only  the  flocculent  silver  magnesium  urate 
is  left.  Rinse  the  rod,  and  centrifuge  the  tube  for  about  2  min- 
utes. Decant,  add  about  5  c.c.  of  water,  then  stir  the  precipitate 
thoroughly  (again  carefully  rinsing  the  stirring  rod),  centrifuge 
(2  minutes)  and  pour  off  the  liquid. 


Add  about  5  c.c.  of  water,  .5  c.c.  of  5  per  cent,  sodium  cyanide 
solution  (poisonous:  j  c.c.  may  be  fatal  dose),  and  I  c.c.  of  uric 
acid  (phenol)  reagent,  and  stir.  A  perfectly  clear  solution  should 
result.  Add  5  c.c.  of  20  per  cent,  sodium  carbonate  solution  and 
allow  the  tube  to  stand  for  20  minutes. 

Measure  10  c.c.  (.5  mg.)  of  standard  uric  acid  solution  and  50 
c.c.  water  into  a  100  c.c.  graduated  flask.  Add  4  c.c.  of  uric  acid 
(phenol)  reagent,  2  c.c.  of  the  sodium  cyanide  solution,  and 
20  c.c.  of  20  per  cent,  sodium  carbonate,  and  let  the  flask  stand 
20  minutes. 

At  the  end  of  20  minutes  centrifuge  for  2  minutes  the  tube 
containing  the  uric  acid  from  blood.  Pour  the  blue  liquid  as  com- 
pletely as  possible  into  a  25  c.c.  graduated  flask,  fill  to  the  mark 
with  water,  and  mix. 

Fill  the  100  c.c.  flask  containing  the  standard  to  the  mark, 
shake,  and  centrifuge  a  portion. 

Read  these  two  solutions  against  each  other  in  the  colorimeter, 
setting  the  standard  at  20  m.m. 

In  making  the  calculations  it  is  to  be  noted  that  half  a  milli- 
gram of  uric  acid  diluted  to  100  c.c.  is  the  standard,  whereas 
the  unknown  represents  3  c.c.  of  blood  diluted  to  25  c.c.  Twenty 
multiplied  by  100,  and  divided  by  (4X2X3)  and  by  the  read- 
ing of  the  unknown,  gives  the  uric  acid  in  milligrams  per  100  c.c. 
of  blood. 

Or :  833  divided  by  the  reading  of  the  unknown  (in  m.m.)  gives 
the  desired  figure. 

Preparation  of  Standard  Uric  Acid  Solution.  —In  a  small  beaker 
weigh  accurately  250  mg.  of  pure  uric  acid,  and  dissolve  by  the  addi- 
tion of  30-40  c.c.  .4  per  cent,  solution  of  lithium  carbonate.  When 
solution  is  complete,  transfer  it  without  loss  of  a  single  drop  to  a 
500  c.c.  volumetric  flask.  Fill  to  the  mark  with  distilled  water  and 
mix. 

Transfer  100  c.c.  of  the  freshly  prepared  uric  acid  solution  to  a 
liter  flask,  add  500-700  c.c.  of  water  and  10  c.c.  normal  hydrochloric 
acid  (or  I  c.c.  concentrated  acid),  and  fill  up  to  the  mark  with 
water.  Transfer  the  solution  to  a  bottle,  add  5  c.c.  of  chloroform, 
and  mix. 

The  acidified  uric  acid  solution  keeps  a  long  time  (several  weeks). 
From  the  fresh  carbonate  solution  of  uric  acid  several  liters. of  the 
dilute  acidified  solution  can  of  course  be  prepared,  but  it  is  not  yet 
known  (Oct.,  1915)  whether  these  solutions  keep  indefinitely. 

Twenty  c.c.  of  the  solution  contains  I  mg.  of  uric  acid 

169 


Preparation  of  Uric  Acid  and  Phenol  Reagent.— Transfer  to 

a  flask  (capacity  about  1500  c.c.)  : 
750  c.c.  of  water, 
100  g.  of  sodium  tungstate, 
20  g.  of  phosphomolybdic  acid, 
50  c.c.  of  phosphoric  acid  (85  per  cent.  H3PO4), 
100  c.c.  of  concentrated  hydrochloric  acid. 

Insert  a  funnel  in  the  flask  and  partly  close  the  opening  of  the 
funnel  with  a  watch  glass.  Boil  the  mixture  gently  for  two  hours. 
A  deep  straw  yellow  solution  should  be  obtained.  It  should  not  turn 
appreciably  blue  when  a  sample,  5  c.c.,  is  rendered  alkaline  with 
sodic  carbonate.  Dilute  to  a  liter. 

Preparation  of  Uric  Acid  Reagent. — Introduce  into  a  flask : 
750  c.c.  of  water, 
100  g.  of  sodium  tungstate, 

80  c.c.  of  phosphoric  acid   (85  per  cent.  H3PO4). 
Partly  close  the  mouth  of  the  flask  with  a  funnel  and  small  watch 
glass  and  boil  gently  for  two  hours.    Dilute  to  a  liter. 

Colorimetric  Method  for  the  Determination  of  Creatinin  in  Blood 

(J.  Biol.  Chem.,  if,  475)- — Transfer  10  c.c.  of  blood  (or  milk) 
to  a  5,0  c.c.  shaking  cylinder  which  can  be  closed  with  a  glass 
stopper.  Fill  up  to  the  50  c.c.  mark  with  saturated  picric  acid 
solution  and  shake  a  few  times.  Add  about  I  g.  dry  picric  acid 
to  the  mixture  and  continue  the  shaking  for  five  minutes.  Trans- 
fer the  mixture  to  centrifuge  tubes,  centrifuge,  and  pour  the 
supernatant  fluid  through  a  filter.  This  is  the  most  economical 
process  where  but  little  blood  is  available.  When  this  is  not  the 
case,  the  quantities  taken  may  be  doubled,  and  the  filtration  can 
then  be  made  without  the  preliminary  centrifuging  process. 

By  the  above  treatment  the  protein  materials  are  removed  and 
the  creatin  and  creatinin  are  obtained  in  the  picric  acid  filtrate. 
The  filtrate  is  at  the  same  time  practically  a  saturated  picric  acid 
solution.  For  the  colorimetric  determination  of  the  preformed 
creatinin  in  the  filtrate  prepare  a  correspondingly  dilute  solution 
of  creatinin  in  saturated  picric  acid  solution.  A  solution  con- 
taining .2  mg.  of  creatinin  per  100  c.c.  is  suitable  for  this  purpose. 
This  can  be  prepared  in  a  few  moments  by  transferring  I  mg.  of 
creatinin  from  the  standard  creatinin  solution  used  for  urine 
work  (see  p.  97)  to  a  500  c.c.  volumetric  flask,  and  then  making 
up  to  volume  with  saturated  picric  acid  solution.  This  solution 

171 


can  be  kept  on  hand,  as  the  creatinin  is  not  precipitated  on  ac- 
count of  the  great  dilution.  Add  the  same  amount  of  alkali  to 
equal  volumes  of  filtrate  from  the  blood  (or  milk)  and  of  the 
known  picric  acid  solution.  The  color  produced  corresponds  to 
the  amount  of  creatinin  present,  provided  that  neither  contains 
more  than  one  and  one-half  times  as  much  as  the  other.  It  is 
absolutely  essential,  however,  that  exactly  the  same  amount  of 
the  10  per  cent,  alkali  should  be  added  to  each  solution,  because 
in  saturated  solutions  of  picric  acid  the  alkali  deepens  the  color 
even  when  no  creatinin  is  present.  The  amount  of  alkali  found 
to  yield  the  most  reliable  results,  when  making  creatinin  determi- 
nations in  this  manner,  is  5  c.c.  of  10  per  cent,  sodium  hydrate 
per  100  c.c.  of  picric  acid  solution. 

When  measuring  out  the  alkali  with  an  ordinary  buret  the 
simplest  way  to  get  the  same  amount  of  alkali  for  the  unknown 
and  the  standard  is  to  determine  how  many  drops,  as  obtained 
from  the  buret,  correspond  to  5  c.c.,  and  then  to  add  one-fifth  of 
that  number  to  20  c.c.  of  the  unknown  filtrate  and  to  20  c.c.  of 
the  known  solution.  Ten  c.c.  of  the  filtrate  from  the  blood  (or 
milk)  may  be  used  for  the  color  comparison,  in  which  case  the 
alkali  added  must  be  only  as  many  drops  as  correspond  to  .5  c.c. 

Sometimes  the  filtrate  obtained  from  blood  becomes  slightly 
turbid  after  the  addition  of  the  alkali.  It  must  then  be  centri- 
fuged  or  filtered  before  using  it  for  the  color  comparison. 

Ten  minutes'  standing  after  the  addition  of  the  alkali  is  ade- 
quate for  the  development  of  the  color,  and  the  solutions  are 
then  ready  for  the  color  comparison  without  any  further  dilution. 
They  are  accordingly  transferred  to  the  cylinders  of  the  Duboscq 
colorimeter  and  compared  in  the  usual  manner.  The  standard 
creatinin  solution  in  this  case  can  advantageously  be  set  at  20  mm. 
because  the  color  of  the  solutions  are  not  very  deep,  but  it  is 
not  at  all  essential  that  this  should  be  done. 

The  calculation  of  the  creatinin  in  the  blood  (or  milk)  is  the 
same  whether  the  standard  is  set  at  10,  15,  or  20  mm.,  and 
whether  10  or  20  c.c.  of  the  filtrate  was  taken  for  the  making  of 
the  color  reaction.  When  10  c.c.  of  blood  is  diluted  to  50  c.c.,  or 
20  c.c.  to  100  c.c.,  and  the  standard  contains  .2  mg.  of  creatinin 
per  100  c.c.,  according  to  the  directions  described  above,  the 
reading  of  the  standard  divided  by  the  reading  of  the  unknown 
gives,  without  any  further  calculations,  the  creatinin  in  milli- 
grams contained  in  100  c.c.  of  blood  (or  milk). 


Colorimetric  Method  for  the  Determination  of  Creatin  plus  Cre- 
atinin  in  Blood  (Milk). — For  the  determination  of  the  so-called 
total  creatinin  in  blood,  milk,  and  exudates,  the  preliminary  pre- 
cipitation with  picric  acid  is  conducted  in  exactly  the  same  man- 
ner as  has  been  described  above  in  connection  with  the  determina- 
tion of  the  preformed  creatinin.  The  filtrate  obtained  from  10  c.c. 
of  blood  diluted  with  picric  acid  solution  to  50  c.c.  amounts  usually 
to  rather  more  than  30  c.c.,  and  10  c.c.  of  this  filtrate  is  all  that  is 
needed  for  the  total  creatinin  determination.  Measure  out  10  c.c. 
of  the  creatin-creatinin  filtrate  into  a  small  Erlenmeyer  flask 
(capacity  25  or  50  c.c.)  or  large  test  tube. 

Cover  the  flask  or  test  tube  containing  the  filtrate  with  tinfoil, 
transfer  to  the  autoclave,  and  heat  to  about  120°  C  for  about 
twenty  minutes.  When  using  the  autoclave,  it  is  important  not 
to  open  it  at  the  end  of  the  heating  until  the  temperature  has 
fallen  to  below  ioc°  so  as  to  avoid  all  mechanical  losses. 

When  cooled  to  room  temperature,  rinse  the  solution  into  a 
25  c.c.  volumetric  flask,  or  a  measuring  cylinder,  with  saturated 
picric  acid  solution,  making  a  volume  of  25  c.c.  Add  1*25  c.c.  of 
10  per  cent,  sodium  hydrate  solution  for  the  development  of  the 
color  reaction. 

Two  standard  creatinin  solutions  in  saturated  picric  acid  are 
necessary  in  this  determination  because  of  the  variations  in  the 
creatin  contents  of  normal  blood.  When  working  on  hospital 
patients,  the  variations  are  greater  still,  and  three  standard  cre- 
atinin determinations  are  desirable.  These  standard  solutions 
contain  .5,  I,  and  2  mg,  of  creatinin,  respectively,  per  100  c.c.  of 
saturated  picric  acid  solution.*  To  20  c.c.  of  each  of  these  solu- 
tions in  measuring  cylinders  add  I  c.c.  of  10  per  cent,  sodium  hy- 
drate. By  inspection  one  can  readily  tell  which  standard  comes 
nearest  to  having  the  same  color  as  the  unknown,  and  with  this 
as  a  standard  make  the  color  comparison  in  the  usual  manner,  by 
the  help  of  the  Duboscq  colorimeter.  The  colors  of  these  solu- 
tions are  much  deeper  than  those  obtained  in  the  determination 
of  the  preformed  creatinin,  and  the  standard  solution  is  conse- 
quently usually  set  at  10  mm. 

The  reading  of  the  standard  in  mm.,  multiplied  by  125  and  by 
.5,  i,  and  2,  according  to  which  standard  is  used,  when  divided 

*  These  solutions  are  made  by  putting  i,  2,  and  4  c.c.  of  the  standard 
creatinin  zinc  chlorid  solution  in  200  c.c.  volumetric  flasks,  and  making 
up  to  volume  with  saturated  picric  acid  solution. 

175 


by  the  reading  of  the  unknown  in  mm.,  gives  the  amount  of  cre- 
atin-creatinin  in  milligrams  per  100  c.c.  of  blood  (or  milk). 

Determination  of  Chlorids  in  Blood,  Exudates,  and  Transudates 

(McLean  and  Van  Slyke:  /.  Biol.  Chem.,  21,  361  [/9/5]). — In 
this  method  the  chlorids  are  precipitated  in  the  presence  of  nitric 
acid  by  standard  silver  nitrate  solution,  the  silver  chlorid  is  re- 
moved by  filtration,  and  the  excess  of  silver  is  titrated  back  with 
standard  potassium  iodid  solution.  The  titration  is  performed  in 
the  presence  of  nitrous  acid  and  starch,  so  that  the  first  drop  of 
iodid  in  excess  of  the  silver  present  is  changed  to  free  iodin  and 
gives  the  blue  starch-iodin  color.  The  optimum  acidity  for  the 
end-point  is  fixed  by  the  addition  of  trisodium  citrate  in  amount 
equivalent  (l/$  molecule)  to  the  free  nitric  acid  present. 
The  solutions  required  are 

I.  An  acid  solution  of  silver  nitrate,  I  c.c.  of  which  is  equiv- 
alent to  2  mg.  of  NaCl.     This  solution  contains  5.812  g.  silver 
nitrate  and  250  c.c.  concentrated  nitric  acid  (sp.  gr.   1.42)  in  one 
liter. 

II.  A  solution  of  potassium  iodid,  I  c.c.  of  which  is  equivalent 
to  i  mg.  of  NaCl.    This  solution  contains  3  g.  of  potassium  iodid 
per  liter. 

III.  A  solution  containing  sodium  citrate,  sodium  nitrite,  and 
starch,  which  substances  respectively  regulate  the  acidity,  pro- 
vide an  oxidizing  agent  for  the  iodid,  and  serve  as  indicator. 
This  solution  contains  446  g.  of  trisodium  citrate,  20  g.  of  sodium 
nitrite,  and  2.5  g.  of  soluble  starch  per  liter. 

First  dissolve  the  starch  in  about  500  c.c.  of  boiling  water.  Add 
the  citrate  and  nitrite,  and  heat  the  mixture  until  all  is  dissolved. 
Filter  the  solution,  while  still  hot,  through  cotton,  wash  the  filter 
with  hot  water,  cool,  and  finally  make  up  to  a  volume  of  1,000  c.c. 

Standardize  solution  II.  against  solution  I.  by  adding  5  c.c. 
of  the  latter  to  5  c.c.  of  solution  III.,  and  titrating  with  solution 
II.  until  the  blue  end-point  is  obtained.  On  the  basis  of  this 
titration,  dilute  the  iodid  solution  to  such  a  degree  that  10  c.c. 
is  exactly  equivalent  to  5  c.c.  of  the  silver  solution. 

As  a  preliminary  step  to  the  titration  of  chlorids  in  blood,  or 
other  albuminous  fluids,  the  protein  must  first  be  removed.  This 
is  accomplished  as  follows:  Run  2  c.c.  of  oxalated  blood  from 
an  Qstwald  pipet  into  10  c.c.  of  10  per  cent,  magnesium  sulphate 
solution  contained  in  a  20  c.c.  volumetric  flask.  Rinse  the  pipet 

.177 


twice  by  drawing  up  into  it  the  solution  from  the  flask.  Add  2 
drops  of  50  per  cent,  acetic  acid,  fill  to  the  mark  with  water, 
invert  a  few  times  to  secure  a  thorough  mixing  of  its  contents, 
and  finally  place  for  10  minutes  in  a  beaker  of  boiling  water. 
After  cooling  pour  the  contents  of  the  flask  onto  about  .3  g.  of 
Merck's  blood  charcoal  in  a  small  beaker.  Let  stand  a  few  min- 
utes and  filter.  A  water  clear  filtrate  should  be  obtained,  which 
is  now  ready  for  titration. 

The  final  titration  of  the  protein  free  filtrate  is  carried  out  as 
follows :  Pipet  10  c.c.  of  filtrate  (equivalent  to  I  c.c.  of  blood) 
into  a  25  c.c.  volumetric  flask,  add  5  c.c.  of  the  silver  nitrate  solu- 
tion, and  fill  to  the  mark  with  distilled  water.  Mix,  allow  to 
stand  five  minutes,  and  filter  through  a  dry  filter  paper.  Pipet 
20  c.c.  of  this  filtrate  into  a  50  c.c.  Erlenmeyer  flask,  add  4  c.c. 
of  solution  III.,  and  then  run  in  the  potassium  iodid  (solution 
II.)  from  a  buret  until  the  end-point  is  reached.  The  first  definite 
blue  color  marks  the  end-point,  and  with  practice  is  easily  de- 
termined. The  results  may  be  calculated  from  the  following 
formula  when  20  c.c.  of  filtrate  from  the  silver  chlorid  is  titrated : 

12.5  (8  —  c.c.  KI  solution  used) 

Grams  NaCl  per  liter 

c.c.  filtrate  (from  charcoal)  used 

Before  beginning  work  it  is  desirable  to  make  sure  by  qualitative 
tests  that  the  chemicals  employed  (more  particularly  the  magnesium 
sulphate,  potassium  nitrite,  and  charcoal)  are  free  from  chlorids. 
Before  making  up  solution  III  it  is  also  well  to  determine  whether 
the  soluble  starch  provided  gives  a  blue  end-point  with  iodin.  Much 
of  the  soluble  starch  on  the  market  is  so  highly  dextrinized  that  it 
gives  a  reddish-brown  color  with  iodin  and  is  therefore  unfit  for 
titration  work. 

Nephelometric  Method  for  the  Determination  of  Fat  in  Blood 

(Bloor:  J.  Biol.  Chem.,  17,  37?) . — Run  3  c.c.  of  blood  slowly  and 
with  shaking  into  a  100  c.c.  volumetric  flask  containing  about  80 
c.c.  of  a  mixture  of  redistilled  alcohol  and  ether  (3:1).  Raise  the 
contents  of  the  flask  just  to  boiling  (with  constant  shaking)  in  a 
water-bath,  cool  in  running  water,  make  up  to  the  100  c.c.  mark 
with  more  alcohol-ether,  mix,  and  filter  into  a  small  flask  or  bottle. 
Stopper  tightly  as  soon  as  filtration  is  finished  to  avoid  loss  of 
liquid  by  evaporation. 

179 


Measure  15  c.c.  of  the  filtrate,  containing  about  2  mg.  of  fat, 
into  a  small  beaker,  add  2  c.c.  of  N  sodium  ethylate,  and  evaporate 
the  mixture  just  to  dryness  on  the  water-bath.  To  the  dry  residue 
add  5  c.c.  of  alcohol-ether  (1:3),  and  warm  gently  until  the 
flakes  of  alkali  are  loosened  from  the  bottom  of  the  beaker.  To 
the  mixture  add  50  c.c.  of  water,  and  stir  until  a  clear  solution 
is  obtained.  Add  5  c.c.  of  a  standard  solution  of  oleic  acid  in  al- 
cohol-ether, containing  about  2  mg.  oleic  acid,  to  50  c.c.  of  water 
in  another  beaker.  To  the  standard,  and  to  the  blood  fat  solution 
add  (as  nearly  simultaneously  as  possible)  10  c.c.  of  10  per  cent, 
hydrochloric  acid.  Allow  the  suspensions  so  produced  to  stand 
for  five  minutes,  and  then  compare  by  means  of  the  Duboscq 
colorimeter  previously  converted  into  a  nephelometer. 

For  the  comparison,  fill  the  two  nephelometer  tubes,  after  rins- 
ing with  the  solutions,  to  the  same  height  (the  meniscus  slightly 
above  the  dark  collar  at  the  top  of  the  tubes),  and  place  in  the 
nephelometer,  with  the  standard  tube  always  on  the  same  side. 
Set  the  movable  jacket  on  the  standard  tube  at  a  convenient  point 
(30  mm.  in  the  modified  colorimeter  described  below),  and  make 
comparisons  by  adjusting  the  jacket  on  the  test  solution  until  the 
images  show  equal  illumination.  Make  five  readings  alternately 
from  above  and  from  below,  and  take  the  average  as  the  reading. 
Make  the  calculations  in  the  same  way  as  in  the  colorimetric 
methods,  the  values  being  inversely  proportional  to  the  read- 
ings. 

Changing  the  Duboscq  Colorimeter  into  a  Nephelometer  (7.  Biol. 
Chem.,  22,  145). — A  simple  method  for  transforming  the  Duboscq 
colorimeter  into  a  nephelometer  is  described  in  the  Journal  of  Bio- 
logical  Chemistry,  Vol.  22,  p.  145,  1915.  The  extra  parts  neces- 
sary are  supplied  in  an  improved  form  by  the  International  In- 
strument Company  of  Cambridge,  Mass.  By  the  use  of  these 
parts  the  change  may  be  quickly  made  as  follows :  Unscrew  the 
movable  glass  prisms  of  the  colorimeter,  slip  the  brass  collars 
for  the  nephelometer  tubes  into  place,  and  fasten  on  the  plate 
from  which  the  prisms  were  removed.  Slip  the  movable  jackets 
into  the  holes  in  the  cup  supports,  and  after  pushing  the  nephelom- 
eter tubes  into  place  in  the  collars,  the  instrument  is  ready  for 
use.  A  darkened  room  and  a  light-tight  box  for  the  light  are 
necessary.  The  box  should  be  about  48  cm.  long,  32  cm.  high, 
and  20  cm.  wide  for  the  ordinary  colorimeter.  It  should  contain 

181 


a  bracket  at  one  end  to  support  the  light  (a  50  watt  "Mazda") 
at  the  height  of  the  nephelometer  tubes,  and  a  stop  at  the  other 
end,  against  which  the  instrument  may  be  pushed  and  so  placed 
that  the  nephelometer  tubes  are  about  30  cm.  from  the  light.  A 
slot  in  the  top  of  the  box  to  receive  the  telescope  of  the  instru- 
ment and  a  dark  curtain  to  cover  the  end  of  the  box  after  the 
instrument  is  pushed  into  place  complete  the  equipment  of  the 
box.  All  exposed  parts  should  be  painted  a  dull  black. 

Since  the  readings  obtained  from  suspensions  of  different 
strength  are  not  exactly  proportional  to  the  amount  of  precipitate 
present,  it  is  necessary  to  calibrate  the  instrument  for  different 
strengths  and  make  corrections  accordingly.  If,  however,  the  so- 
lution to  be  tested  is  within  25  per  cent,  of  the  value  of  the  stand- 
ard, no  correction  is  necessary. 

A  Method  for  the  Determination  of  Cholesterin  in  Blood  or  Blood 
Serum  (Bloor,  not  yet  published).— The  method  consists  in  the 
application  of  the  Autenrieth-Funk  procedure  (Autenrieth  and 
Funk — Milnch.  med.  Wochenschr.,  1913,  Vol.  69,  p.  1243)  to  the 
alcohol-ether  extract  of  blood  or  serum  prepared  as  for  the  de- 
termination of  fat. 

Measure  10  c.c.  of  the  extract  into  a  small  beaker,  and  evaporate 
just  to  dryness  on  the  water-bath  or  electric  stove.  (Any  heating 
after  dryness  is  reached  produces  a  brownish  color,  which  makes 
the  determination  difficult  or  impossible.) 

Extract  the  cholesterin  from  the  dry  residue  by  boiling  out  3 
or  4  times  with  small  portions  (2-3  c.c.)  of  chloroform  and  de- 
canting. Evaporate  the  combined  extracts  to  a  little  less  than 
5  c.c.,  transfer  to  a  10  c.c.  graduated  cylinder,  and  make  the  vol- 
ume up  to  5  c.c.  A  little  turbidity  does  not  matter,  since  it  dis- 
appears on  adding  the  reagents.  Measure  5  c.c.  of  a  standard 
cholesterin  solution  in  chloroform,  containing  .5  mg.  of  choles- 
terin, into  a  similar  10  c.c.  graduate.  Add  to  each  2  c.c.  of  acetic 
anhydrid  and  .1  c.c.  of  concentrated  H2SO4.  Mix  the  solutions  by 
inverting  two  or  three  times,  and  set  the  cylinders  in  the  dark 
for  15  minutes ;  then  transfer  the  solutions  to  the  colorimeter  cups, 
and  compare  as  usual,  setting  the  standard  at  15  m.m. 

The  cement  of  the  colo'rimeter  cups  must,  of  course,  not  be  soluble 
in  chloroform.  Plaster-of-Paris  has  been  found  satisfactory,  or  even 
ordinary  glue,  if  the  cups  are  not  used  for  any  other  purpose. 

183 


Method  for  the  Determination  of  Sugar  in  Blood  (Lewis  and 
Benedict,  J.  Biol.  Chem.,  20,  61,  1915). — Pipet  2  c.c.  of  oxalated 
blood  into  a  25  c.c.  volumetric  flask  containing  5  c.c.  of  distilled 
water ;  the  water  and  blood  are  mixed  in  order  to  insure  complete 
hemolysis  of  the  latter.  Add  15  c.c.  of  saturated  picric  acid  solu- 
tion, fill  up  to  the  mark  with  water,  and  shake. 

Filter,  and  measure  two  8  c.c.  portions  of  the  filtrate  into  large 
Jena  test  tubes  (22  X  120  m.m.)  for  duplicate  determinations. 
Add  2  c.c.  of  saturated  picric  acid  solution  and  exactly  I  c.c.  of 
10  per  cent,  sodium  carbonate  solution  (as  well  as  two  glass 
beads  or  pebbles,  and  two  or  three  drops  of  kerosene).  Evap- 
orate the  contents  of  the  test  tubes  rapidly  over  a  free  flame  until 
precipitation  occurs.  Add  about  3  c.c.  of  water,  and  heat  the  tube 
again  to  boiling  so  as  to  dissolve  the  precipitate.  Now  transfer 
the  contents  of  the  tube  quantitatively  to  a  10  c.c.  volumetric 
flask,  cool,  fill  up  to  the  mark  with  water,  shake,  then  filter  through 
cotton  into  the  colorimeter  chamber.  Compare  the  color  at  once 
with  that  obtained  from  £4  mg.  of  dextrose,  5  c.c.  of  saturated 
picric  acid,  and  I  c.c.  of  saturated  sodium  carbonate,  after  the 
solution  of  these  substances  has  been  evaporated  over  a  free  flame 
and  diluted  to  10  c.c.  in  the  same  way  as  in  the  case  of  the  un- 
known. 

Instead  of  the  dextrose  standard  a  permanent  standard  may  be 
used  which  consists  of  .064  mg.  picramic  acid  and  .1  g.  sodium 
carbonate  (anhydrous)  in  1,000  c.c.  Before  being  used  this  solu- 
tion should  be  standardized  against  pure  dextrose. 

To  calculate  the  results  the  following  formula  may  be  used: 

reading  of  standard    mg.  dextrose  in  stand- 
ard 

Mg.  dextrose  per  c.c.  of  blood  = 

reading  of  unknown         c.c.  of  blood  used 

But  as  the  amount  of  dextrose  in  the  standard  is  .64  mg.  and  the 
amount  of  blood  used  is  .64  c.c.,  the  second  fraction  equals  unity, 
and  the  equation  is : 

reading  of  standard 
Mg.  dextrose  per  c.c.  of  blood  =  — 

reading  of  unknown 

The  per  cent,  of  blood  sugar  is,  of  course,  one  tenth  of  the 
figure  thus  obtained. 

THE   END 

'185  • 


INDEX 


Aceto  acetic  acid  in  urine,  133 
Acetone  in  urine,  125,  131 
Acidimetry,   I 
Acidity  of  urine,  determination  of, 

103 

Acids,  strong  and  weak,  13 
Albumine    in    urine,    determination 

of,  145-147 

Albumins,  tests  for,  67-72 
Alkalimetry,    I 
Amino  acids,  75-77 
Ammonia,  determination  of,  17,  79, 

81,  83 
Ammonia    in   blood,    determination 

of,  165 
Atom  weights,  3 

Benedict's    method    for    determina- 
tion of  sugar  in  blood,  185 
method     for     determination     of 

sugar  in  urine,  49 
reagent  for  sugar,  43 
standard  copper  solution,  49 
Beta  oxybutyric  acid  in  urine,  135- 

139 

Bile,  119 
Blood,  qualitative  experiments  with, 

109-110 
Bloor's     method     for     determining 

cholesterin  in  blood,  183 
nephelometric  method  for  fats  in 

blood,  179 
Bone,   117 

Carbohydrates,  39-59 

Calcium  in  urine,  determination  of, 

149 

Catalysis,  25 
Chlorids,  in  urine,  determination  of, 

103 
method  for  determining  in  blood, 

177 


Cholesterin,   Liebermann's   reaction 

for,  37 
method  for  determining  in  blood, 

183 

Colloids,  61-63 
Creatin,  in  urine,  97 
method  for  determining  in  blood, 

175 
Creatinin,  95 

determination   of,   in  urine,  97 
method  for  determining  in  blood, 

171 
Cystin,  preparation  of,  77 

Dextrin,  57 

Diacetic  acid  in  urine,  133 

Diastase,  27 

Fat,  3-38 

emulsification  of,  37 

method  for  determining  in  blood, 
179 

digestion,  35 
Fats,  iodin  number  of,  31 

saponification,  33 
Fatty  acids,  preparation  of,  33 

solubility   of,   33 

titration  of,  35 

Gelatin,  73 
Globulin,  test  for,  73 
Glycerin,  aerolein  test  for,  35 
Glycogen,  57 

Hippuric  acid,  99 

in  urine,  determination  of,  143 
Hydrogen-ion       concentration       in 

urine,  151-156 
Hydrochloric  acid,  test  for,  15 

Indican  in  urine,  105 
Indicators,  5,   13 
Invert  sugar,  55 


187 


Keratin,  73 

Lactic  acid,  test  for,  15 
Lecithin,   37 
Leucin,  75 
Levulose,  test  for,  47 

Magnesium  in  urine,  determination 

of,  149 

Maltose,  preparation  of,  55 
Marshall's  method  for  urea  deter- 
mination, 89 

method  for  urea  in  blood,  163 
Mass  Law,  29 
McCrudden's    method    for    calcium 

and  magnesium,  149 
Metabolism  experiments,  105 
Milk,    113 

sugar,  preparation  of,  55 
sugar  determination,  113 

Nephelometer,  181 
Nessler's  reagent,  161 
Nitrogen,    non-protein,    determina- 
tion of  in  blood,  159 
test  for,  in  protein,  67 
determination,  colorimetric  meth- 
od  for,    83 

in  ammonium  salts,  17 
Kjeldahl's  method,  17,  21,  23 

Pentoses,  test  for,  47 

Pepsin,  25 

Peptones,  73 

Phenol      determination     in     urine, 

141 

Phenol-uric  acid  reagent,  171 
Phosphates  in  urine,  101 
Phosphoproteins,   73 
Phosphorus,  test  for  in  protein,  67 
Potassium   in  urine,   determination 

of,  149 

Proteins,  61-77 
Proteoses,  73 

Reversible  reaction,  29 

Shaffer's   method   for  beta  oxybu- 

tyric  acid  in  urine,  137 
Sodium  in  urine,  determination  of, 

149 


Solution,  equivalent,  I 

Fehling's,  41 

normal,  i 

standard  acetone,  127-129 

standard  ammonia,  85 

standard  creatinin,  97 

standard  hydrochloric  acid,  n 

standard  iodin,  127 

standard  oxalic  acid,  7 

standard  protein,  147 

standard  sodic  hydrate,  9 

standard  sodium  thiosulphate,  12 

standard  uric  acid,  169 
Solutions,  colloidal,   61 
Sugar,  Benedict's  reagent  for,  43 

Benedict's  test  for,  41 

bismuth   test    for,   45 

Fehling's  test  for,  41 

fermentation  test  for,  49 

method  for  determining,  in  blooc 
185 

phenylhydrazin  test  for,  45 

Selivanoff's  test  for,  47 

Trommer's  test  for,  39 
Sugar      determination,      Benedict' 
method,  49 

polariscope  method,  51 
Starch  paste,  preparation  of,  57 
Sulphates,      determination     of      ii 

urine,  99 
Sulphur,  test  for  in  protein,  67 

total,  determination  of   in  urine 
101 

Trypsin,  27 
Tyrosin,  75 

Urea,     colorimetric     determinatioi 
of,     in  urine,  87-89 

in  blood,  determination  of,  161-163 

reactions  with,  85 
Uric  acid  in  urine,  colorimetric  de- 
termination, 93 

murexid  test  for,  91 

phosphotungstic  acid  test  for,  9] 

preparation  from  urine,  89 

titration  of,  91 
Uric  acid  reagent,  171 
Urine,  79-107,   125-157 


188 


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